Cell culture media and methods of antibody production

ABSTRACT

Cell culture media are provided herein as are methods of using the media for cell culture and antibody production from cells. Compositions comprising antibodies and fragments thereof, produced by the methods herein are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2014/029758, filed Mar. 14, 2014, which claims the prioritybenefit of U.S. Provisional Application Ser. No. 61/801,247, filed Mar.15, 2013, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to cell culture media for use in culturinga mammalian cell comprising a nucleic acid encoding bevacizumab, or afragment thereof, and to methods of using the media in bevacizumabproduction as well as compositions and kits comprising the bevacizumab,or a fragment thereof, produced by the methods provided herein.

BACKGROUND OF THE INVENTION

Cell culture manufacturing technology is widely used for the productionof protein-based therapeutics, such as antibodies, for use inpharmaceutical formulations. Commercial production of protein-basedproducts, such as an antibody product, requires optimization of cellculture parameters in order for the cell to produce enough of theprotein product to meet manufacturing demands. However, when cellculture parameters are optimized for improving productivity of theprotein product it is also necessary to maintain the desired qualityattributes of the product such as the glycosylation profile, aggregatelevels, charge heterogeneity, and amino acid sequence integrity (Li, etal., 2010, mAbs., 2(5):466-477).

Bevacizumab, also known as “Avastin®”, is a recombinant humanizedmonoclonal antibody that binds vascular endothelial growth factor in invitro and in vivo assay systems (U.S. Pat. Nos. 7,227,004; 6,884,879;7,060,269; 7,169,901; 7,297,334) and is used in the treatment of cancer,where it inhibits tumor growth by blocking the formation of new bloodvessels. Bevacizumab has an approximate molecular weight of 149,000daltons, is glycosylated, and is produced in a mammalian cell (ChineseHamster Ovary) expression system in a nutrient cell culture medium.

Improved and cost-effective methods of producing bevacizumab, or afragment thereof, are desirable. Cell culture media comprisingcomponents that enable a cell to produce a desired amount ofbevacizumab, or a fragment thereof, while maintaining acceptable productquality attributes of bevacizumab, or a fragment thereof, would bebeneficial. Cell culture media for use in producing manufacturing-scaleamounts of bevacizumab, or a fragment thereof, would be particularlyadvantageous.

BRIEF SUMMARY OF THE INVENTION

The invention provided herein discloses, inter alia, methods ofproducing bevacizumab, or a fragment thereof, in a cell culture mediumcomprising at least two of copper, insulin, and cystine, and optionallycomprises an animal-derived hydrolysate and/or a plant-derivedhydrolysate. Also provided are methods for culturing a mammalian cell(e.g., a CHO cell) comprising a nucleic acid encoding bevacizumab, or afragment thereof, using a cell culture medium provided herein. Furtherdisclosed herein are cell culture media compositions that enhance theamount (e.g., enhance the titer) of bevacizumab, or a fragment thereof,produced from a mammalian cell in cell culture, as well as compositionscomprising bevacizumab, or a fragment thereof, produced by the methodsdescribed herein.

Accordingly, in one aspect, the invention provides a method of producingbevacizumab, or a fragment thereof, comprising the step of culturing amammalian cell comprising a nucleic acid encoding bevacizumab orfragment thereof in a cell culture medium, wherein the cell culturemedium comprises two or more components selected from the groupconsisting of copper, insulin, and cystine, and wherein the cellproduces bevacizumab, or a fragment thereof. In a further embodiment,the cell culture medium comprises copper and insulin. In another furtherembodiment, the cell culture medium comprises copper and cystine. In yetanother further embodiment, the cell culture medium comprises insulinand cystine. In still another further embodiment, the cell culturemedium comprises copper, insulin, and cystine. In any of the embodimentsherein, the cell culture medium can further comprise a plant-derivedhydrolysate, an animal-derived hydrolysate or both a plant-derivedhydrolysate and an animal-derived hydrolysate. In some of theembodiments herein, the cell culture medium comprises copper at aconcentration selected from the concentrations listed in Table 1. Insome of the embodiments herein, the cell culture medium comprisesinsulin at a concentration selected from the concentrations listed inTable 1. In some of the embodiments herein, the cell culture mediumcomprises cystine at a concentration selected from the concentrationslisted in Table 1. It is understood that any combination of amounts ofcopper, insulin and/or cystine, e.g., the amounts provided in Table 1,are intended the same as if each and every combination of amounts werespecifically and individually listed. In any of the embodiments herein,the cell culture medium can comprise insulin at a concentration of fromabout 7.0 mg/L to about 11.0 mg/L. In any of the embodiments herein, thecell culture medium can comprise copper at a concentration of from about69.0 nM to about 400.0 nM. In any of the embodiments herein, the cellculture medium can comprise cystine at a concentration of from about 0.8mM to about 2.5 mM. In any of the embodiments herein, the cell culturemedium can comprise an animal-derived hydrolysate at a concentration offrom about 5.6 g/L to about 38.0 g/L. In any of the embodiments herein,the cell culture medium can comprise a plant-derived hydrolysate at aconcentration of from about 1.4 g/L to about 6.2 g/L. In someembodiments herein, the cell culture medium is a basal cell culturemedium. In some embodiments herein, the cell culture medium is a feedcell culture medium. In some embodiments herein, the cell culture mediumis a basal cell culture medium comprising at least one of copper,insulin, and cystine, and where the basal cell culture medium issupplemented (e.g., at a period of time following initiation of a cellculture cycle, such as any one of at least one time, two times, at leastthree times, at least four times, at least five times, at least sixtimes, at least seven times, etc. of a cell culture cycle) with a feedcell culture medium comprising any one or more of insulin, ananimal-derived hydrolysate and a plant-derived hydrolysate. In anothervariation, a feed cell culture medium comprises any one or more ofinsulin, an animal-derived hydrolysate, a plant-derived hydrolysate,cysteine and cystine. In another variation, a feed cell culture mediumcomprises insulin, an animal-derived hydrolysate, a plant-derivedhydrolysate and cysteine. In another variation, a feed cell culturemedium comprises any one or more of insulin, an animal-derivedhydrolysate, a plant-derived hydrolysate and cystine. The feed cellculture medium may comprise the any one or more of insulin, ananimal-derived hydrolysate, a plant-derived hydrolysate, cysteine andcystine in any amount provided herein. In some embodiments herein, thecell culture medium comprises insulin and the method further comprisesthe step of adding an additional amount of insulin to the cell culturemedium (e.g., such as via a feed medium introduced to the basal cellculture medium at a period of time following initiation of the cellculture cycle). In some embodiments, the additional amount of insulin isadded in an amount to provide insulin in the cell culture medium at aconcentration selected from the concentrations listed in Table 1. In afurther embodiment, the additional amount of insulin is added to thecell culture medium at least once during the cell culture cycle. Inanother further embodiment, the additional amount of insulin is added tothe cell culture medium at least three times during the cell culturecycle. In yet another further embodiment, the additional amount ofinsulin is added to the cell culture medium at least six times duringthe cell culture cycle. In some of the embodiments herein, theadditional amount of insulin is added in an amount to provide insulin inthe cell culture medium at a concentration of from about 5.6 mg/L toabout 66.0 mg/L. In some of the embodiments herein, the method furthercomprises the step of adding an additional amount of animal-derivedhydrolysate and/or plant-derived hydrolysate to the cell culture mediumprovided herein (e.g., such as via a feed medium introduced to the basalcell culture medium at a period of time following initiation of the cellculture cycle). In some of the embodiments herein, the additional amountof animal-derived hydrolysate and/or plant-derived hydrolysate added tothe cell culture is added in an amount to provide animal-derivedhydrolysate and/or plant-derived hydrolysate in the cell culture mediumat a concentration selected from the concentrations listed in Table 1.In any of the embodiments herein, the cell (e.g., a CHO cell capable ofproducing bevacizumab, or a fragment thereof) can be cultured at atemperature ranging from about 28° C. to about 37° C. or about 31° C. toabout 37° C. In any of the embodiments herein, bevacizumab, or afragment thereof, can be secreted into the cell culture medium. In anyof the embodiments herein, the method can further comprise the step ofrecovering the bevacizumab, or a fragment thereof, from the cellculture. In a particular variation, the recovered bevacizumab ispurified.

Also provided herein are methods of producing bevacizumab, or a fragmentthereof, in a cell culture medium comprising an animal-derivedhydrolysate and a plant-derived hydrolysate and optionally furthercomprising copper, insulin and/or cystine. In one such aspect, the ananimal-derived hydrolysate is present in a greater amount than theplant-derived hydrolysate. In one such variation, the animal-derivedhydrolysate is present in the cell culture media at a concentration offrom about 5.6 g/L to about 38.0 g/L or from about 7.0 g/L to about 35.0g/L or from about 7.0 g/L to about 25.0 g/L or from about 7.0 g/L toabout 15.0 g/L or from about 8.0 g/L to about 12.0 g/L or from about 7.0g/L to about 11.0 g/L or about any one of 5 g/L, 10 g/L, 15 g/L, 20 g/L,25 g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L or 50 g/L or about any one of 5g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, or 12 g/L or about 10g/L. In another variation, the plant-derived hydrolysate is present inthe cell culture media at a concentration of from about 1.4 g/L to about6.2 g/L or from about 1.5 g/L to about 5.5 g/L or from about 1.5 g/L toabout 4.5 g/L or from about 1.5 g/L to about 3.5 g/L or from about 1.5g/L to about 2.5 g/L or from about 1.75 g/L to about 2.75 g/L or fromabout 2.0 g/L to about 3.0 g/L or from about 2.25 g/L to about 2.75 g/Lor about any one of 1.75 g/L, 2.0 g/L, 2.25 g/L, 2.5 g/L, 3.0 g/L, 3.25,3.5 g/L, 3.75 g/L, or 4.0 g/L or about any one of 2.0 g/L, 2.25 g/L, 2.5g/L or 3.0 g/L or about 2.5 g/L. It is understood that each and everycombination of amount of animal-derived hydrolysate and plant-derivedhydrolysate is described the same as if each and every combination werespecifically and individually listed.

In some aspects, the invention provides bevacizumab, or fragmentthereof, produced by any of the methods described herein.

In other aspects, the invention provides a composition comprising: (i)bevacizumab, or a fragment thereof, produced by any of the methodsdescribed herein and (ii) a pharmaceutically acceptable carrier.

In some aspects, the invention also provides a method of culturing amammalian cell comprising a nucleic acid encoding bevacizumab, or afragment thereof, the method comprising the step of contacting themammalian cell with a cell culture medium comprising two or morecomponents selected from the group consisting of copper, insulin andcystine. In a further embodiment, the cell culture medium comprisescopper and insulin. In another further embodiment, the cell culturemedium comprises copper and cystine. In yet another further embodiment,the cell culture medium comprises insulin and cystine. In still yetanother further embodiment, the cell culture medium comprises copper,insulin, and cystine. In some embodiments herein, the cell culturemedium further comprises a plant-derived hydrolysate, an animal-derivedhydrolysate or both a plant-derived hydrolysate and an animal-derivedhydrolysate. In some of the embodiments herein, the cell culture mediumcomprises insulin at a concentration of from about 7.0 mg/L to about11.0 mg/L. In some of the embodiments herein, the cell culture mediumcomprises copper at a concentration of from about 69.0 nM to about 400.0nM. In some of the embodiments herein, the cell culture medium comprisescystine at a concentration of from about 0.8 mM to about 2.5 mM. In someof the embodiments herein the cell culture medium comprises ananimal-derived hydrolysate at a concentration of from about 5.6 g/L toabout 38.0 g/L. In some of the embodiments herein, the cell culturemedium comprises a plant-derived hydrolysate at a concentration of fromabout 1.4 g/L to about 6.2 g/L. In some of the embodiments herein, thecell culture medium is a basal cell culture medium. In some embodimentsherein, the cell culture medium is a feed cell culture medium. In someembodiments herein, the cell culture medium is a basal cell culturemedium comprising at least one of copper, insulin, and cystine, andwhere the basal cell culture medium is supplemented (e.g., at a periodof time following initiation of a cell culture cycle, such as any one ofat least two times, at least three times, at least four times, at leastfive times, at least six times, at least seven times, etc. of a cellculture cycle) with a feed cell culture medium comprising any one ormore of insulin, an animal-derived hydrolysate and a plant-derivedhydrolysate. In some embodiments herein, the cell culture mediumcomprises insulin and the method further comprises the step of adding anadditional amount of insulin to the cell culture medium. In a furtherembodiment, the additional amount of insulin is added to the cellculture medium at least once during the cell culture cycle. In anotherfurther embodiment, the additional amount of insulin is added to thecell culture medium at least three times during the cell culture cycle.In yet another further embodiment, the additional amount of insulin isadded to the cell culture medium at least six times during the cellculture cycle. In some embodiments, the additional amount of insulin isadded in an amount to provide insulin in the cell culture medium at aconcentration of from about 5.6 mg/L to about 66.0 mg/L. In some of theembodiments herein, the method further comprises the step of adding anadditional amount of animal-derived hydrolysate and/or plant-derivedhydrolysate to the cell culture medium provided herein (e.g., such asvia a feed medium introduced to the basal cell culture medium at aperiod of time following initiation of the cell culture cycle). In someof the embodiments herein, the additional amount of animal-derivedhydrolysate and/or plant-derived hydrolysate added to the cell cultureis added in an amount to provide animal-derived hydrolysate and/orplant-derived hydrolysate in the cell culture medium at a concentrationselected from the concentrations listed in Table 1. In some of theembodiments herein, the cell is cultured at a temperature ranging about28° C. to about 37° C. or from about 31° C. to about 37° C. In any ofthe embodiments herein, bevacizumab, or a fragment thereof, can besecreted into the cell culture medium. In some embodiments herein, themammalian cell is contacted with the cell culture medium during thecell's growth phase. In some embodiments herein, the mammalian cell iscontacted with the cell culture medium during the cell's productionphase.

In other aspects, the invention provides a kit for supplementing a cellculture medium for use in culturing a mammalian cell comprising anucleic acid encoding bevacizumab, or a fragment thereof, the kitcomprising at least two of components (i)-(iii): (i) insulin in anamount to provide from about 7.0 mg/L to about 11.0 mg/L insulin in thecell culture medium; (ii) cystine in an amount to provide from about 0.8mM to about 2.5 mM cystine in the cell culture medium; (iii) and copperin an amount to provide from about 25.0 nM to about 400.0 nM copper inthe cell culture medium. In some embodiments, the kit further comprisesa plant-derived hydrolysate. In a further embodiment, the kit comprisesthe plant-derived hydrolysate in an amount to provide from about 1.4 g/Lto about 6.2 g/L plant-derived hydrolysate in the cell culture medium.In any of the embodiments herein, the kit can further comprise ananimal-derived hydrolysate. In some embodiments, the kit comprises theanimal-derived hydrolysate in an amount to provide from about 5.6 g/L toabout 38.0 g/L animal-derived hydrolysate in the cell culture medium.The kit may additionally contain instructions for use, such asinstructions for use in supplementing a cell culture medium.

In another aspect, the invention provides a cell culture medium for usein culturing a mammalian cell comprising a nucleic acid encodingbevacizumab, or a fragment thereof, the cell culture medium comprisingat least two of components (i)-(iii): (i) from about 7.0 mg/L to about11.0 mg/L insulin; (ii) from about 25.0 nM to about 400.0 nM copper; and(iii) from about 0.8 mM to about 2.5 mM cystine. In some embodiments,the cell culture medium comprises from about 7.0 mg/L to about 11.0 mg/Linsulin; and from about 25.0 nM to about 400.0 nM copper. In someembodiments, the cell culture medium comprises: from about 7.0 mg/L toabout 11.0 mg/L insulin; and from about 0.8 mM to about 2.5 mM cystine.In some embodiments, the cell culture medium comprises from about 25.0nM to about 400.0 nM copper; and from about 0.8 mM to about 2.5 mMcystine. In any of the embodiments herein, the cell culture medium canfurther comprise from about 1.4 g/L to about 6.2 g/L plant-derivedhydrolysate. In any of the embodiments herein, the cell culture mediumcan further comprise from about 5.6 g/L to about 38.0 g/L animal-derivedhydrolysate.

In yet another aspect, the invention also provides a compositioncomprising (a) a mammalian cell comprising a nucleic acid encodingbevacizumab, or a fragment thereof; and (b) any cell culture mediumprovided herein.

In another aspect, the invention provides a composition comprising: (a)bevacizumab, or a fragment thereof; and (b) any cell culture mediumprovided herein. In a further embodiment, bevacizumab, or a fragmentthereof, is secreted into the medium by a mammalian cell comprising anucleic acid encoding bevacizumab, or a fragment thereof.

In some aspects, also provided herein is a method of enhancing titer ofbevacizumab, or a fragment thereof, from a mammalian cell comprising anucleic acid encoding bevacizumab, or a fragment thereof, the methodcomprising the step of culturing the mammalian cell in a cell culturemedium comprising at least two of insulin, copper and cystine, whereintiter is enhanced relative to culturing the mammalian cell in a cellculture medium without at least two of insulin, copper and cystine. Insome embodiments, the cell culture medium comprises copper and insulin.In some embodiments, the cell culture medium comprises copper andcystine. In some embodiments, the cell culture medium comprises insulinand cystine. In some embodiments, the cell culture medium comprisescopper, insulin, and cystine. In any of the embodiments herein, the cellculture medium can further comprise a plant-derived hydrolysate, ananimal-derived hydrolysate or both a plant-derived hydrolysate and ananimal-derived hydrolysate. In any of the embodiments herein, the cellculture medium can comprise insulin at a concentration of from about 7.0mg/L to about 11.0 mg/L. In any of the embodiments herein, the cellculture medium can comprise copper at a concentration of from about 69.0nM to about 400.0 nM. In any of the embodiments herein, the cell culturemedium can comprise cystine at a concentration of from about 0.8 mM toabout 2.5 mM. In any of the embodiments herein, the cell culture mediumcan comprise an animal-derived hydrolysate at a concentration of fromabout 5.6 g/L to about 38.0 g/L. In any of the embodiments herein, thecell culture medium can comprise a plant-derived hydrolysate at aconcentration of from about 1.4 g/L to about 6.2 g/L. In someembodiments, the cell culture medium is a basal cell culture medium. Insome embodiments herein, the cell culture medium is a feed cell culturemedium. In some embodiments herein, the cell culture medium is a basalcell culture medium comprising at least one of copper, insulin, andcystine, and where the basal cell culture medium is supplemented (e.g.,at a period of time following initiation of a cell culture cycle, suchas any one of at least two times, at least three times, at least fourtimes, at least five times, at least six times, at least seven times,etc. of a cell culture cycle) with a feed cell culture medium comprisingany one or more of insulin, an animal-derived hydrolysate and aplant-derived hydrolysate. In some embodiments, the cell culture mediumcomprises insulin and the method further comprises the step of adding anadditional amount of insulin to the cell culture medium. In a furtherembodiment, the additional amount of insulin is added to the cellculture medium at least once during the cell culture cycle. In anotherfurther embodiment, the additional amount of insulin is added to thecell culture medium at least three times during the cell culture cycle.In yet another further embodiment, the additional amount of insulin isadded to the cell culture medium at least six times during the cellculture cycle. In some embodiments herein, the additional amount ofinsulin is added in an amount to provide insulin in the cell culturemedium at a concentration of from about 5.6 mg/L to about 66.0 mg/L. Insome of the embodiments herein, the method further comprises the step ofadding an additional amount of animal-derived hydrolysate and/orplant-derived hydrolysate to the cell culture medium provided herein(e.g., such as via a feed medium introduced to the basal cell culturemedium at a period of time following initiation of the cell culturecycle). In some of the embodiments herein, the additional amount ofanimal-derived hydrolysate and/or plant-derived hydrolysate added to thecell culture is added in an amount to provide animal-derived hydrolysateand/or plant-derived hydrolysate in the cell culture medium at aconcentration selected from the concentrations listed in Table 1. In anyof the embodiments herein, the cell may be cultured at a temperatureranging from about 28° C. to about 37° C. or from about 31° C. to about37° C. In any of the embodiments herein, bevacizumab, or a fragmentthereof, can be secreted into the cell culture medium. In any of theembodiments herein, the method may further comprise the step ofrecovering the bevacizumab, or a fragment thereof, from the cellculture. In a further aspect, the recovered bevacizumab, or a fragmentthereof, is purified.

In another aspect, the invention herein provides a method of culturing amammalian cell comprising a nucleic acid encoding bevacizumab, or afragment thereof, in a cell culture medium comprising at least two ofinsulin, copper and cystine, wherein titer of bevacizumab, or a fragmentthereof, is enhanced relative to culturing the mammalian cell in a cellculture medium without at least two of insulin, copper and cystine. Insome embodiments, the cell culture medium comprises copper and insulin.In some embodiments, the cell culture medium comprises copper andcystine. In some embodiments, the cell culture medium comprises insulinand cystine. In some embodiments, the cell culture medium comprisescopper, insulin, and cystine. In some of the embodiments herein, thecell culture medium further comprises a plant-derived hydrolysate, ananimal-derived hydrolysate or both a plant-derived hydrolysate and ananimal-derived hydrolysate. In some of the embodiments herein, the cellculture medium comprises insulin at a concentration of from about 7.0mg/L to about 11.0 mg/L. In some of the embodiments herein, the cellculture medium comprises copper at a concentration of from about 69.0 nMto about 400.0 nM. In some of the embodiments herein, the cell culturemedium comprises cystine at a concentration of from about 0.8 mM toabout 2.5 mM. In some of the embodiments herein, the cell culture mediumcomprises an animal-derived hydrolysate at a concentration of from about5.6 g/L to about 38.0 g/L. In some of the embodiments herein, the cellculture medium comprises a plant-derived hydrolysate at a concentrationof from about 1.4 g/L to about 6.2 g/L. In some embodiments, the cellculture medium is a basal cell culture medium. In some embodimentsherein, the cell culture medium is a feed cell culture medium. In someembodiments herein, the cell culture medium is a basal cell culturemedium comprising at least one of copper, insulin, and cystine, andwhere the basal cell culture medium is supplemented (e.g., at a periodof time following initiation of a cell culture cycle, such as any one ofat least two times, at least three times, at least four times, at leastfive times, at least six times, at least seven times, etc. of a cellculture cycle) with a feed cell culture medium comprising any one ormore of insulin, an animal-derived hydrolysate and a plant-derivedhydrolysate. In any of the embodiments herein, the cell culture mediumcan comprise insulin and the method can further comprise the step ofadding an additional amount of insulin to the cell culture medium. In afurther embodiment, the additional amount of insulin is added to thecell culture medium at least once during the cell culture cycle. Inanother further embodiment, the additional amount of insulin is added tothe cell culture medium at least three times during the cell culturecycle. In yet another further embodiment, the additional amount ofinsulin is added to the cell culture medium at least six times duringthe cell culture cycle. In some of the embodiments herein, theadditional amount of insulin is added in an amount to provide insulin inthe cell culture medium at a concentration of from about 5.6 mg/L toabout 66.0 mg/L. In some of the embodiments herein, the method furthercomprises the step of adding an additional amount of animal-derivedhydrolysate and/or plant-derived hydrolysate to the cell culture mediumprovided herein (e.g., such as via a feed medium introduced to the basalcell culture medium at a period of time following initiation of the cellculture cycle). In some of the embodiments herein, the additional amountof animal-derived hydrolysate and/or plant-derived hydrolysate added tothe cell culture is added in an amount to provide animal-derivedhydrolysate and/or plant-derived hydrolysate in the cell culture mediumat a concentration selected from the concentrations listed in Table 1.In some of the embodiments herein, the cell is cultured at a temperatureranging from about 28° C. to about 37° C. or from about 31° C. to about37° C., such as a temperature of about 31° C., 33° C. or 35° C. It isunderstood that the temperature may vary (either up or down) throughoutthe cell culture process, e.g., within a temperature ranging from about28° C. to about 37° C. In one aspect, the cell is cultured at a firsttemperature of about 35° C. for a first period of time (such as about1-10 or 1-8 or 1-7 days), is cultured at a second temperature of about33° C. for a second period of time (such as about 1-5 or 1-4 or 1-3 or1-2 days), and is cultured at a third temperature of about 31° C. for athird period of time (such as about 1-5 or 1-4 or 1-3 or 1-2 days). Inany of the embodiments herein, bevacizumab, or a fragment thereof, canbe secreted into the cell culture medium. In some embodiments, themethod further comprises the step of recovering the bevacizumab, or afragment thereof, from the cell culture. In one aspect, the recoveredbevacizumab, or a fragment thereof, is purified.

In another aspect, the invention provides a method of producingbevacizumab or a fragment thereof, comprising a step of culturing amammalian cell (e.g., a CHO cell) comprising a nucleic acid encodingbevacizumab or a fragment thereof in a cell culture medium, whereininitial cell culture medium in a cell culture cycle comprises two ormore components selected from the group consisting of copper at aconcentration of from about 69 nM to about 1,000 nM, insulin at aconcentration of from about 1.0 mg/L to about 100.0 mg/L, and cystine ata concentration of from about 0.7 mM to about 2.0 mM, and wherein thecell produces bevacizumab or the fragment. In some embodiments, theinitial cell culture medium comprises (1) copper and insulin; (2) copperand cystine; (3) insulin and cystine; or (4) copper, insulin, andcystine. In some embodiments, the initial cell culture medium comprisesinsulin at a concentration of from about 10.0 mg/L to about 50.0 mg/L.In some embodiments, the initial cell culture medium comprises insulinat a concentration of from about 10.0 mg/L to about 20.0 mg/L. In someembodiments, the initial cell culture medium comprises insulin at aconcentration of about any one of 10.0 mg/L, 15 mg/L, 20 mg/L, and 25mg/L. In some embodiments, the initial cell culture medium comprisescopper at a concentration of from about 325 nM to about 375 nM. In someembodiments, the initial cell culture medium comprises copper at aconcentration of from about 325 nM to about 350 nM. In some embodiments,the initial cell culture medium comprises copper at a concentration ofabout any one of 330 nM, 335 nM, 339 nM, 340 nM, 345 nM and 350 nM. Insome embodiments, the initial cell culture medium comprises cystine at aconcentration of from about 0.7 mM to about 2.0 mM. In some embodiments,the initial cell culture medium comprises cystine at a concentration offrom about 1.0 mM to about 1.6 mM. In some embodiments, the initial cellculture medium comprises cystine at a concentration of about any one of1.0 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM and 1.6 mM. In some embodiments,the initial cell culture medium comprises an animal-derived hydrolysateand/or a plant-derived hydrolysate. In some embodiments, the initialcell culture medium comprises an animal-derived hydrolysate at aconcentration of from about 6.0 g/L to about 20.0 g/L. In someembodiments, the initial cell culture medium comprises an animal-derivedhydrolysate at a concentration of from about 8.0 g/L to about 12.0 g/L.In some embodiments, the initial cell culture medium comprises ananimal-derived hydrolysate at a concentration of from about 9.0 g/L toabout 11.0 g/L. In some embodiments, the initial cell culture mediumcomprises an animal-derived hydrolysate at a concentration of about 13g/L. In some embodiments, the initial cell culture medium comprises aplant-derived hydrolysate at a concentration of from about 1.0 g/L toabout 10.0 g/L. In some embodiments, the cell culture medium comprises aplant-derived hydrolysate at a concentration of from about 2.0 g/L toabout 3.0 g/L. In some embodiments, the cell culture medium comprises aplant-derived hydrolysate at a concentration of from about 2.25 g/L toabout 2.75 g/L. In some embodiments, the initial cell culture mediumcomprises a plant-derived hydrolysate at a concentration of about 2.5g/L. In some embodiments, the cell culture medium comprises both ananimal-derived hydrolysate and a plant-derived hydrolysate, and whereinthe animal-derived hydrolysate is present in a greater amount than theplant-derived hydrolysate. In some embodiments, the initial cell culturemedium comprises insulin and the method further comprises a step ofadding an additional amount of insulin to the cell culture medium duringthe cell culture cycle. In some embodiments, the additional amount ofinsulin is added to the cell culture medium at least once, at leasttwice, at least three times, at least four times, at least five times,or at least six times during the cell culture cycle. In someembodiments, the insulin added each time is from about 5 mg/L to about25 mg/L. In some embodiments, the insulin added each time is about anyone of 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, and 25 mg/L. In someembodiments, the cumulative amount of insulin added during the cellculture cycle is from about 20 mg/L to about 100 mg/L. In someembodiments, the cumulative amount of insulin added during the cellculture cycle is about any one of 20 mg/L, 25 mg/L, 30 mg/L, 35 mg/L, 40mg/L, 45 mg/L, 50 mg/L, 55 mg/L, 60 mg/L, 65 mg/L, 70 mg/L, 75 mg/L, 80mg/L, 85 mg/L, 90 mg/L, 95 mg/L, and 100 mg/L. In some embodiments, theinitial cell culture medium comprises cystine and the method furthercomprises a step of adding an additional amount of cystine to the cellculture medium during the cell culture cycle. In some embodiments,cystine is added in an amount to provide from about 0.1 mM to about 1.5mM additional cystine in the cell culture medium. In some embodiments,cystine is added in an amount to provide about 0.4 mM to about 0.7 mM(e.g., about 0.4 mM to about 0.6 mM, about 0.4 mM to about 0.5 mM)additional cystine in the cell culture medium. In some embodiments,cystine is added in a batch feed during the cell culture cycle. In someembodiments, the method further comprises at least one batch feed duringthe cell culture cycle. In some embodiments, the one batch feed is onday 3 (e.g., in a 14-day cell culture cycle). In some embodiments, themethod comprises two, three, or four batch feeds during the cell culturecycle. In some embodiments, the method comprises a first batch feed onday 3 and a second batch feed on day 6 (e.g., in a 14 day cell culturecycle). In some embodiments, the batch feed medium comprises ananimal-derived hydrolysate and/or a plant-derived hydrolysate. In someembodiments, during the cell culture cycle, the temperature of themedium is reduced by at least about 2, at least about 3, at least about4, or at least about 5 degrees C. relative to the temperature at thebeginning of the culturing. In some embodiments, the temperature of themedium is reduced at least once or at least twice during the cellculture cycle. In some embodiments, the temperature is reduced on day 8and day 10 after the beginning of the culturing. In some embodiments,the cell is cultured at a temperature ranging from about 31° C. to about35° C. In some embodiments, the cell is cultured at a first temperatureof about 35° C. for a first period of time, is cultured at a secondtemperature of about 33° C. for a second period of time, and is culturedat a third temperature of about 31° C. for a third period of time. Insome embodiments, the cell is cultured in the medium having a pH atabout 7.0 to about 7.3. In some embodiments, the method comprises (a)culturing the cell in an initial cell culture medium comprising about 10mg/L insulin, about 325 nM to about 350 nM copper, and about 1.3 mMcystine; (b) providing a first batch feed and an insulin feed to thecell culture medium to provide additional insulin at a concentration ofabout 15 mg/L on day 3 after the beginning of the culturing; and (c)providing a second batch feed comprising cystine to the cell culturemedium to provide additional cystine at a concentration of about 0.4 mMto about 0.7 mM on day 6 after the beginning of the culturing; whereinthe cell is cultured at an initial temperature of about 35° C., and thetemperature is reduced to about 33° C. on day 8 and is further reducedto about 31° C. on day 10 after the beginning of the culturing. In someembodiments, the cell culture cycle is a 14-day cell culture cycle. Insome embodiments, bevacizumab or a fragment thereof is secreted into thecell culture medium. In some embodiments, the method further comprises astep of recovering the bevacizumab or a fragment thereof from the cellculture. In another aspect, the invention provides bevacizumab or afragment thereof produced by any of the methods described above. Inanother aspect, the invention provides a composition comprising: (i)bevacizumab or a fragment thereof produced by any of the methodsdescribed above and (ii) a pharmaceutically acceptable carrier. It is tobe understood that one, some, or all of the properties of the variousembodiments described herein may be combined to form other embodimentsof the present invention.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B are a series of graphs showing the increase inantibody titer yield from bevacizumab producing CHO cells cultured incell culture medium supplemented with insulin. FIG. 1A and FIG. 1B showantibody titers in experiments of six different cell culture protocolsassessed during a 14 day cell culture of CHO cells producingbevacizumab. Antibody production expressed as titer (mg/L). FIG. 1Ashows the results of one batch feed on day 3. FIG. 1B shows the resultsof one batch feed on day 3 and a second batch feed on day 6.

FIG. 2A and FIG. 2B are a series of graphs demonstrating the effect oncell biomass accumulation and antibody production by cell culturesprovided with one or two batch feeds during a 14 day cell culture cycleas a relationship to cell age. FIG. 2A shows cell biomass accumulationin bevacizumab producing CHO cells provided with one batch feed on day 3as compared to bevacizumab producing CHO cells provided with one batchfeed on day 3 and a second batch feed on day 6. p<0.004* for one feedprocess; p<0.018* for two feed process. FIG. 2B shows antibodyproduction from bevacizumab producing CHO cells provided with one batchfeed on day 3 as compared to bevacizumab producing CHO cells providedwith one batch feed on day 3 and a second batch feed on day 6. p<0.042*for one feed process; p<0.82 for two feed process. * indicatessignificant value. “1” indicates cells fed with one batch feed on day 3;“2” indicates cells fed with one batch feed on day 3 and a second batchfeed on day 6.

FIG. 3 is a graph demonstrating the increase in antibody yield frombevacizumab producing CHO cells grown in a new cell culture process.Protocol 1 (triangles) indicates a cell culture process that isdifferent from the cell culture process of Protocol 2; Protocol 2(diamonds) indicates the new cell culture process.

DETAILED DESCRIPTION

Improved and cost-effective methods of producing bevacizumab, or afragment thereof, are provided. Cell culture media comprising componentsthat enable a cell to consistently produce a desired amount ofbevacizumab, or a fragment thereof, while maintaining acceptable productquality attributes of bevacizumab, or a fragment thereof, are described.The cell culture media provided herein may find use in producingmanufacturing-scale amounts of bevacizumab, or a fragment thereof.

The methods provided herein, including: (i) a method of producingbevacizumab, or a fragment thereof; (ii) a method of culturing amammalian cell comprising a nucleic acid encoding bevacizumab, or afragment thereof; and (iii) a method of enhancing production ofbevacizumab, or a fragment thereof, (e.g., enhancing titer yields ofbevacizumab, or a fragment thereof) from a mammalian cell comprising anucleic acid encoding bevacizumab, or a fragment thereof, in one aspectutilize a cell culture medium comprising two or more of copper, insulin,and cystine (such as a cell culture medium comprising copper andinsulin, or a cell culture medium comprising copper and cystine, or acell culture medium comprising insulin and cystine, or a cell culturemedium comprising copper, insulin and cystine). Bevacizumab, or fragmentthereof, produced by any of the methods detailed herein is alsoprovided, as are compositions comprising bevacizumab, or fragmentthereof. In one aspect, bevacizumab, or fragment thereof, produced byany of the methods detailed herein exhibits acceptable product qualityattributes of bevacizumab, or a fragment thereof, such asN-glycosylation profile, charge heterogeneity, and sequence integrity.In a particular variation, the product quality attributes ofbevacizumab, or a fragment thereof, are acceptable if they aresubstantially similar to bevacizumab, or a fragment thereof produced bya method that does not use a cell culture medium comprising at least twoof copper, insulin, and cystine. A cell culture medium comprising two ormore of copper, insulin, and cystine (e.g., a cell culture mediumcomprising: (i) copper and insulin; (ii) copper and cystine; (iii)insulin and cystine; or (iv) copper, insulin and cystine) is alsoprovided. In one variation, the cell culture medium comprising two ormore of copper, insulin, and cystine enhances production of bevacizumab,or a fragment thereof, (e.g., enhances titer yields of bevacizumab, or afragment thereof) by a mammalian cell cultured in the medium relative toculturing the mammalian cell in a cell culture medium without at leasttwo of insulin, copper and cystine. Also provided herein is acomposition comprising a cell culture medium comprising at least two ofcopper, insulin, and cystine and (i) a mammalian cell comprising anucleic acid encoding bevacizumab, or a fragment thereof and/or (ii)bevacizumab, or a fragment thereof. A culturing vessel comprising any ofthe cell culture media provided herein are provided. In one aspect, theculturing vessel is a manufacturing scale culturing vessel, such as aculturing vessel capable of containing at least 2 liters, at least 10liters, at least 100 liters, at least 500 liters, at least 1,000 liters,at least 2,500 liters, at least 5,000 liters, at least 7,500 liters, atleast 10,000 liters, at least 12,000 liters or more of a cell culturemedium provided herein as is required for producing manufacturing scaleamounts of bevacizumab from cell culture. Thus, the methods providedherein may find use in a manufacturing-scale production of bevacizumab,or a fragment thereof.

I. Definitions

The term “bevacizumab” refers to a recombinant humanized anti-VEGFmonoclonal antibody generated according to Presta et al. (1997) CancerRes. 57:4593-4599, also known as “rhuMAb VEGF” or “AVASTIN®”. Itcomprises mutated human IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-human VEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.bevacizumab binds to the same epitope as the monoclonal anti-VEGFantibody A4.6.1 produced by hybridoma ATCC HB 10709. Bevacizumab andother humanized anti-VEGF antibodies are further described in U.S. Pat.No. 6,884,879.

The terms “medium” and “cell culture medium” refer to a nutrient sourceused for growing or maintaining cells. As is understood by a person ofskill in the art, the nutrient source may contain components required bythe cell for growth and/or survival or may contain components that aidin cell growth and/or survival. Vitamins, essential or non-essentialamino acids (e.g., cysteine and cystine), and trace elements (e.g.,copper) are examples of medium components. Any media provided herein mayalso be supplemented with any one or more of insulin, plant hydrolysatesand animal hydrolysates.

“Culturing” a cell refers to contacting a cell with a cell culturemedium under conditions suitable to the survival and/or growth and/orproliferation of the cell.

“Batch culture” refers to a culture in which all components for cellculturing (including the cells and all culture nutrients) are suppliedto the culturing vessel at the start of the culturing process.

“Fed batch cell culture,” as used herein refers to a batch culturewherein the cells and culture medium are supplied to the culturingvessel initially, and additional culture nutrients are fed, continuouslyor in discrete increments, to the culture during the culturing process,with or without periodic cell and/or product harvest before terminationof culture.

“Perfusion culture” is a culture by which the cells are restrained inthe culture by, e.g., filtration, encapsulation, anchoring tomicrocarriers, etc., and the culture medium is continuously orintermittently introduced and removed from the culturing vessel.

“Culturing vessel” refers to a container used for culturing a cell. Theculturing vessel can be of any size so long as it is useful for theculturing of cells.

The term “titer” as used herein refers to the total amount ofrecombinantly expressed antibody produced by a cell culture divided by agiven amount of medium volume. Titer is typically expressed in units ofmilligrams of antibody per milliliter of medium. Titer can be expressedor assessed in terms of a relative measurement, such as a percentageincrease in titer as compared obtaining the protein product underdifferent culture conditions.

A “nucleic acid” refers to polymers of nucleotides of any length, andinclude DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a polymer by DNA or RNApolymerase, or by a synthetic reaction. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and their analogs.If present, modification to the nucleotide structure may be impartedbefore or after assembly of the polymer.

An “isolated nucleic acid” means and encompasses a non-naturallyoccurring, recombinant or a naturally occurring sequence outside of orseparated from its usual context. An isolated nucleic acid molecule isother than in the form or setting in which it is found in nature.Isolated nucleic acid molecules therefore are distinguished from thenucleic acid molecule as it exists in natural cells. However, anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the protein where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

An “isolated” protein (e.g., an isolated antibody) is one which has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with research, diagnostic ortherapeutic uses for the protein, and may include enzymes, hormones, andother proteinaceous or nonproteinaceous solutes. Isolated proteinincludes the protein in situ within recombinant cells since at least onecomponent of the protein's natural environment will not be present.Ordinarily, however, isolated protein will be prepared by at least onepurification step.

A “purified” protein (e.g., antibody) means that the protein has beenincreased in purity, such that it exists in a form that is more purethan it exists in its natural environment and/or when initially producedand/or synthesized and/or amplified under laboratory conditions. Purityis a relative term and does not necessarily mean absolute purity.

“Contaminants” refer to materials that are different from the desiredprotein product (e.g., different from an antibody product). Acontaminant may include, without limitation: host cell materials, suchas CHOP; nucleic acid; a variant, fragment, aggregate or derivative ofthe desired protein; another polypeptide; endotoxin; viral contaminant;cell culture media components, etc.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity. An antibody can be human,humanized and/or affinity matured.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen. The Fab fragment contains the heavy- and light-chain variabledomains and also contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxyterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. Fab′-SH is the designation herein forFab′ in which the cysteine residue(s) of the constant domains bear afree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315,1994.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the invention may be made by avariety of techniques, including, for example, the hybridoma method(e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al.,Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S. Pat. No.5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg etal., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994);Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger,Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32  H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile.

“Pharmaceutically acceptable” carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed (Remington's PharmaceuticalSciences (20^(th) edition), ed. A. Gennaro, 2000, Lippincott, Williams &Wilkins, Philadelphia, Pa.). Often the physiologically acceptablecarrier is an aqueous pH buffered solution. Examples of physiologicallyacceptable carriers include buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween™, polyethylene glycol (PEG), and Pluronics™.

A “sterile” formulation is aseptic or free or essentially free from allliving microorganisms and their spores.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a compound”optionally includes a combination of two or more such compounds, and thelike.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

II. Cell Culture Media

Cell culture media provided herein may find use in methods (e.g., amethod of producing bevacizumab, or a fragment thereof; a method ofculturing a mammalian cell comprising a nucleic acid encodingbevacizumab, or a fragment thereof; and/or a method of enhancingproduction of bevacizumab, or a fragment thereof, such as by enhancingtiter yields of bevacizumab, from a mammalian cell comprising a nucleicacid encoding bevacizumab) and in compositions (e.g., a compositioncomprising a cell culture medium comprising at least two of copper,insulin, and cystine and (i) a mammalian cell comprising a nucleic acidencoding bevacizumab, or a fragment thereof and/or (ii) bevacizumab, ora fragment thereof) as detailed herein.

In some aspects, the cell culture medium provided herein comprisescomponents (e.g., at least two of copper, insulin, and cystine) that maybe used in culturing a cell that produces bevacizumab, or a fragmentthereof, wherein the cell, when cultured in the presence of the mediacomponents (e.g., at least two of copper, insulin, and cystine),produces bevacizumab, or a fragment thereof, in a desired amount, whichmay be in an amount that is greater than the amount of bevacizumabproduced by a cell cultured in a cell culture medium that does notcontain the media components (e.g., a cell culture medium that does notcontain at least two of copper, insulin, and cystine). In one aspect,the cell culture media provided herein is used in culturing a cell thatproduces bevacizumab, or a fragment thereof, wherein the cell, whencultured in the presence of the media components (e.g., at least two ofcopper, insulin, and cystine), produces bevacizumab, or a fragmentthereof, in a desired amount and with an acceptable quality attribute,such as an acceptable molecular weight. As used herein, “an acceptablequality attribute” of bevacizumab can refer to a chemical and/orphysical attribute required for regulatory approval or marketing ofbevacizumab and may be the chemical and/or physical attribute used inassessing lot-to-lot consistency of batches of bevacizumab, or afragment thereof, produced by a cell.

In other aspects of the invention, cell culture media components (e.g.,at least two of copper, insulin, and cystine) have been identified ascapable of providing antibody-producing cells with improved oracceptable quality attributes that contribute to higher production ofbevacizumab (e.g., results in higher titer of bevacizumab) as comparedto cells that produce bevacizumab and are cultured in a cell culturemedium that does not contain these components (a cell culture mediumthat does not contain at least two of copper, insulin and cystine).Certain identified media components (e.g., at least two of copper,insulin, and cystine) can be used to provide an antibody-producing cell(e.g., a CHO cell) with the capability of producing bevacizumab, or afragment thereof, with an acceptable titer, which in one aspect is atiter greater than the titer obtained when the cells producebevacizumab, or a fragment thereof, in a cell culture medium that doesnot comprise at least two of copper, insulin, and cystine. As usedherein, “an acceptable titer” of an antibody produced from a culturedcell (e.g., bevacizumab produced from a CHO cell) can as a non-limitingexample refer to the amount of antibody required to meetmanufacturing-scale production of the antibody or to the amount ofantibody required to assess consistency in lot-to-lot batches of theantibody product. The cell culture media provided herein may improve theamount of bevacizumab that is produced from a cell comprising a nucleicacid encoding bevacizumab as compared to the amount of bevacizumabproduced from the cell cultured in a different media.

A cell culture medium for use in culturing a cell for use in culturing amammalian cell comprising a nucleic acid encoding bevacizumab, or afragment thereof, is provided, wherein the cell culture medium comprisesany one or more of: (a) copper; (b) insulin; (c) cystine; (d) ananimal-derived hydrolysate; and (e) a plant-derived hydrolysate. In someembodiments, the cell culture medium comprises 2 or 3 or 4 or 5 ofcomponents (a), (b), (c), (d) and (e). It is understood that the cellculture medium provided herein may contain any combination of components(a), (b), (c), (d) and (e) the same as if each and every combinationwere specifically and individually listed. For example, it is understoodthat a cell culture medium comprising three of components (a), (b), (c),(d) and (e) may comprise any combination of the components so long as atleast three of the components are present (e.g., a cell culture mediumcomprising components (a), (b) and (c) or comprising components (a), (d)and (e) or comprising components (c), (d) and (e) are contemplated). Insome embodiments, a cell culture medium provided herein comprisescomponents (a), (b), (c), (d) and (e). In some embodiments, a cellculture medium provided herein comprises (a) and (b). In someembodiments, a cell culture medium provided herein comprises (a) and(c). In some embodiments, a cell culture medium provided hereincomprises (b) and (c).

In some aspects, a cell culture medium as provided herein contains oneor more media components selected from the group consisting of copper,insulin, cystine in amounts as described in Table 1. In someembodiments, the cell culture medium further comprises an animal-derivedhydrolysate in amounts as described in Table 1. In other embodiments,the cell culture medium further comprises a plant-derived hydrolysate inamounts as described in Table 1. In some embodiments, the cell culturemedium further comprises both an animal-derived hydrolysate and aplant-derived hydrolysate in amounts as described in Table 1.

It is also understood that a cell culture medium provided herein maycomprise any one or more of the cell culture medium components of Table1 (any one or more of copper, insulin, cystine, an animal-derivedhydrolysate and a plant-derived hydrolysate) in any of the amountslisted in Table 1, the same as if each and every combination ofcomponents and amounts were specifically and individually listed. In onevariation, the cell culture medium provided herein comprises two orthree or four or each of copper, insulin, cystine, an animal-derivedhydrolysate and a plant-derived hydrolysate in any of the amounts listedin Table 1, the same as if each and every combination of components andamounts were specifically and individually listed. In one aspect, thecell culture medium comprises at least two of copper, insulin andcystine in any of the amounts listed in Table 1, and an in furthervariation further comprises an animal-derived hydrolysate and/or aplant-derived hydrolysate in any of the amounts listed in Table 1.

TABLE 1 Exemplary Amounts of Media Components Component Amount ofComponent in Medium (a) Insulin from about 1.0 mg/L to about 100.0 mg/L;from about 5.0 mg/L to about 80.0 mg/L; from about 5.0 mg/L to about60.0 mg/L; from about 5.0 mg/L to about 50.0 mg/L; from about 5.0 mg/Lto about 40.0 mg/L; from about 5.0 mg/L to about 30.0 mg/L; from about5.0 mg/L to about 25.0 mg/L; from about 10.0 mg/L to about 25.0 mg/L;from about 10.0 mg/L to about 30.0 mg/L; from about 15.0 mg/L to about20.0 mg/L; from about 5.0 mg/L to about 15.0 mg/L; from about 6.0 mg/Lto about 12.0 mg/L; from about 7.0 mg/L to about 11.0 mg/L; from about8.0 mg/L to about 10.0 mg/L; from about 10 mg/L to about 100 mg/L; fromabout 10 mg/L to about 50 mg/L; from about 10 mg/L to about 35 mg/L;from about 10 mg/L to about 250 mg/L; from about 1.0 mg/L to about 66mg/L; from about 1.0 mg/L to about 60 mg/L; from about 1.0 mg/L to about50 mg/L; from about 1.0 mg/L to about 40 mg/L; from about 1.0 mg/L toabout 30 mg/L; from about 1.0 mg/L to about 20 mg/L; from about 1.0 mg/Lto about 10 mg/L; from about 10 mg/L to about 66 mg/L; from about 20mg/L to about 66 mg/L; from about 30 mg/L to about 66 mg/L; from about40 mg/L to about 66 mg/L; from about 50 mg/L to about 66 mg/L; fromabout 60 mg/L to about 66 mg/L; from about 5.6 mg/L to about 66 mg/L;from about 10 mg/L to about 60 mg/L; from about 20 mg/L to about 50mg/L; from about 30 mg/L to about 40 mg/L; from about 1 mg/L to about 14mg/L; from about 1.3 mg/L to about 13 mg/L; from about 1.6 mg/L to about12 mg/L; from about 1.4 mg/L to about 11 mg/L; from about 5.6 mg/L toabout 14 mg/L; from about 5.9 mg/L to about 13 mg/L; from about 6.2 mg/Lto about 12 mg/L; from about 7 mg/L to about 11 mg/L; about any of 1.0or 2.0 or 3.0 or 4.0 or 5.0 or 6.0 or 7.0 or 8.0 or 9.0 or 10 or 11 or12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or24 or 25 or 26 or 27 or 27 or 28 or 29 or 30 or 31 or 32 or 33 or 34 or35 or 36 or 37 or 38 or 39 or 40 or 41 or 42 or 43 or 45 mg/L; any of5.6 mg/L, 6 mg/L, 6.2 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 11 mg/L, 12mg/L, 13 mg/L, 14 mg/L or 15 mg/L or 16 mg/L or 17 mg/L or 18 mg/L or 19mg/L or 20 mg/L or 21 mg/L or 22 mg/L or 23 mg/L or 24 mg/L or 25 mg/L;at least about any of 1.0 or 3.0 or 5.0 or 7.0 or 10 or 11 or 12 or 13or 14 or 15 or 16 or 17 or 18 or 19 or 20 mg/L and no more than about 44or 24 or 14 or 11 mg/L. (b) Cystine from about 0.5 mM to about 2.5 mM;from about 0.5 mM to about 2.0 mM; from about 0.5 mM to about 1.75 mM;from about 0.5 mM to about 2.5 mM; from about 0.8 mM to about 2.5 mM;from about 0.8 mM to about 2.25 mM; from about 0.8 mM to about 2.0 mM;from about 0.8 mM to about 1.75 mM; from about 0.8 mM to about 1.6 mM;from about 0.8 mM to about 1.25 mM; from about 0.8 mM to about 1.0 mM;from about 1.0 mM to about 1.6 mM; from about 1.0 mM to about 2.5 mM;from about 1.25 mM to about 2.5 mM; from about 1.5 mM to about 2.5 mM;from about 1.75 mM to about 2.5 mM; from about 2.0 mM to about 2.5 mM;from about 2.25 mM to about 2.5 mM; from about 0.9 mM to about 2.0 mM;from about 0.8 mM to about 1.75 mM; from about 0.9 mM to about 1.5 mM;from about 1.0 mM to about 1.25 mM; from about 1.0 mM to about 2.0 mM;from about 1.0 mM to about 1.5 mM; from about 1.2 mM to about 1.4 mM;about any of 0.8 or 0.9 or 1.0 or 1.1 or 1.2 or 1.3 or 1.4 or 1.5 or 1.6mM; any of 0.8 mM, 0.85 mM, 0.9 mM, 0.95 mM, 1.0 mM, 1.05 mM, 1.1 mM,1.15 mM, 1.2 mM, 1.25 mM, 1.3 mM, 1.35 mM, 1.4 mM, 1.5 mM, 1.55 mM, 1.6mM, 1.65 mM, 1.7 mM, or 1.75 mM; at least about any of 0.8 or 0.9 or 1.0or 1.1 mM and no more than about 1.75 or 1.6 or 1.5 or 1.4 mM. (c)Copper from about 69 nM to about 1,000.0 nM; from about 20 nM to about480.0 nM; from about 20 nM to about 400 nM; from about 20 nM to about350 nM; from about 20 nM to about 300 nM; from about 20 nM to about 250nM; from about 20 nM to about 200 nM; from about 20 nM to about 150 nM;from about 20 nM to about 100 nM; from about 20 nM to about 50 nM; fromabout 50 nM to about 480 nM; from about 100 nM to about 480 nM; fromabout 150 nM to about 480 nM; from about 200 nM to about 480 nM; fromabout 250 nM to about 480 nM; from about 300 nM to about 480 nM; fromabout 325 nM to about 375 nM; from about 350 nM to about 480 nM; fromabout 400 nM to about 480 nM; from about 50 nM to about 450 nM; fromabout 100 nM to about 400 nM; from about 150 nM to about 350 nM; fromabout 200 nM to about 300 nM; from about 22 nM to about 440 nM; fromabout 26 nM to about 400 nM; from about 30 nM to about 360 nM; fromabout 54 nM to about 480 nM; from about 62 nM to about 440 nM; fromabout 69 nM to about 400 nM; from about 80 nM to about 400 nM; fromabout 100 nM to about 400 nM; from about 125 nM to about 400 nM; fromabout 150 nM to about 400 nM; from about 200 nM to about 400 nM; fromabout 250 nM to about 400 nM; from about 300 nM to about 400 nM; fromabout 325 nM to about 375 nM; from about 325 nM to about 350 nM; any ofabout 25 or 26 or 27 or 28 or 29 or 30 or 40 or 50 or 60 or 69 or 100 or110 or 120 or 125 or 130 or 140 or 150 or 160 or 170 or 175 or 180 or190 or 200 or 210 or 220 or 225 or 230 or 240 or 250 or 260 or 270 or275 or 280 or 290 or 300 or 310 or 320 or 325 or 330 or 335 or 336 or337 or 338 or 339 or 340 or 345 or 350 or 360 or 370 or 375 or 380 or390 or 400 nM; any of 54 nM, 56 nM, 58 nM, 60 nM, 62 nM, 64 nM, 66 nM,68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 nM, 75 nM, 100 nM, 125 nM,150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM,375 nM, or 400 nM; at least any of about 20 or 25 or 30 or 35 or 40 or45 or 50 or 55 or 60 or 65 or 70 or 80 nM and no more than about 420 or400 or 380 or 360 nM. (d) Animal- from about 6.0 g/L to about 20 g/L;from about 5.6 g/L to about 38 derived g/L; from about 5.6 g/L to about30 g/L; from about 5.6 g/L to about hydrolysate 25 g/L; from about 5.6g/L to about 20 g/L; from about 7.0 g/L to about 20 g/L; from about 9.0to about 11.0 g/L; from about 5.6 g/L to about 10 g/L; from about 5.6 toabout 38 g/L; from about 10 g/L to about 38 g/L; from about 15 g/L toabout 38 g/L; from about 20 g/L to about 38 g/L; from about 25 g/L toabout 38 g/L; from about 30 g/L to about 38 g/L; from about 35 g/L toabout 38 g/L; from about 10 g/L to about 30 g/L; from about 15 g/L toabout 25 g/L; from about 5.6 g/L to about 14 g/L; from about 5.9 g/L toabout 13 g/L; from about 6.2 g/L to about 12 g/L; from about 7.0 g/L toabout 11.0 g/L; from about 7.0 g/L to about 35.0 g/L; from about 7.0 g/Lto about 25.0 g/L; from about 7.0 g/L to about 15.0 g/L; from about 8.0g/L to about 12.0 g/L; any of about 2.8 or 3.0 or 3.2 or 3.4 or 3.6 or3.8 or 4.0 or 4.2 or 4.4 or 4.6 or 4.8 or 5.0 or 5.2 or 5.4 or 5.6 or 6or 6.2 or 7 or 7.4 or 7.8 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15or 20 or 25 or 30 or 35 or 40 or 45 or 50 g/L; any of 2.8 g/L, 3.0 g/L,3.2 g/L, 3.4 g/L, 3.6 g/L, 3.8 g/L, 4.0 g/L, 4.2 g/L, 4.4 g/L, 4.6 g/L,4.8 g/L, 5.0 g/L or 6 g/L, 6.2 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L,12 g/L, 13 g/L, or 14 g/L; at least any of about 2.8 or 3.2 or 3.4 or3.6 or 3.8 or 4.0 or 4.2 or 4.6 or 5.0 or 5.6 or 6 or 6.2 or 7 or 8 or 9g/L and no more than about 14 or 13 or 12 or 11 g/L. (e) Plant-derivedfrom about 1.0 g/L to about 10.0 g/L; from about 1.4 g/L to about 6.2hydrolysate g/L; from about 1.4 g/L to about 6.0 g/L; from about 1.4 g/Lto about 5.5 g/L; from about 1.4 g/L to about 5.0 g/L; from about 1.4g/L to about 4.5 g/L; from about 1.4 g/L to about 4.0 g/L; from about1.4 g/L to about 3.5 g/L; from about 1.4 g/L to about 3.0 g/L; fromabout 1.4 g/L to about 2.5 g/L; from about 1.4 g/L to about 2.0 g/L;from about 2.0 g/L to about 6.2 g/L; from about 2.5 g/L to about 6.2g/L; from about 3.0 g/L to about 6.2 g/L; from about 3.5 g/L to about6.2 g/L; from about 4.0 g/L to about 6.2 g/L; from about 4.5 g/L toabout 6.2 g/L; from about 5.0 g/L to about 6.2 g/L; from about 5.5 g/Lto about 6.2 g/L; from about 1.5 g/L to about 6.0 g/L; from about 2.0g/L to about 5.5 g/L; from about 2.5 g/L to about 5.0 g/L; from about3.0 g/L to about 4.5 g/L; from about 3.0 g/L to about 3.2 g/L; fromabout 3.5 g/L to about 4.0 g/L; from about 1.4 g/L to about 3.4 g/L;from about 1.5 g/L to about 3.0 g/L; from about 1.75 g/L to about 2.8g/L; from about 1.5 g/L to about 5.5 g/L; from about 1.5 g/L to about4.5 g/L; from about 1.5 g/L to about 3.5 g/L; from about 1.5 g/L toabout 2.5 g/L from about 1.75 g/L to about 2.75 g/L; from about 2.0 g/Lto about 3.0 g/L; from about 2.25 g/L to about 2.75 g/L; any of about1.4 or 1.5 or 1.75 or 2.0 or 2.25 or 2.5 or 2.8 or 3.0 or 3.1 or 3.25 or3.4 or 3.5 or 3.75 or 4.0 g/L; any of 1.4 g/L, 1.5 g/L, 1.75 g/L, 2.0g/L, 2.25 g/L, 2.5 g/L, 2.8 g/L, 3.0 g/L, 3.2 g/L, 3.25 g/L, 3.4, 3.75,or 4.0 g/L; at least any of about 1.4 or 1.5 or 1.75 g/L and no morethan about 3.4 or 3 or 2.8 g/L.

In one variation, insulin is present in the cell culture media at aconcentration of from about 1.0 mg/L to about 100.0 mg/L or from about5.0 mg/L to about 80.0 mg/L or from about 5.0 mg/L to about 60.0 mg/L orfrom about 5.0 mg/L to about 50.0 mg/L or from about 5.0 mg/L to about40.0 mg/L or from about 5.0 mg/L to about 30.0 mg/L or from about 5.0mg/L to about 25.0 mg/L or from about 10.0 mg/L to about 25.0 mg/L orfrom about 10.0 mg/L to about 30.0 mg/L or from about 15.0 mg/L to about20.0 mg/L or from about 5.0 mg/L to about 15.0 mg/L or from about 6.0mg/L to about 12.0 mg/L or from about 7.0 mg/L to about 11.0 mg/L orfrom about 8.0 mg/L to about 10.0 mg/L or at a concentration of aboutany one of 5.0 mg/L, 6.0 mg/L, 7.0 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L,11.0 mg/L, 12.0 mg/L, 13.0 mg/L, 14.0 mg/L, 15.0 mg/L, 16.0 mg/L, 17.0mg/L, 18.0 mg/L, 19.0 mg/L, 20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L,24.0 mg/L, 25.0 mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0mg/L or at a concentration of about any one of 7 mg/L, 8.0 mg/L, 9.0mg/L, 10.0 mg/L or 11.0 mg/L.

In one variation, copper is present in the cell culture media at aconcentration of from about 69.0 nM to about 400.0 nM or from about 80nM to about 400 nM or from about 100 nM to about 400 nM or from about125 nM to about 400 nM or from about 150 nM to about 400 nM or fromabout 200 nM to about 400 nM or from about 250 nM to about 400 nM orfrom about 300 nM to about 400 nM or from about 325 nM to about 375 nMor from about 325 nM to about 350 nM or at a concentration of about anyone of 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM,300 nM, 325 nM, 350 nM, 375 nM or 400 nM or at a concentration of aboutany one of 330 nM, 335 nM, 340 nM, 345 nM or 350 nM or at aconcentration of about 335 nM, 336 nM, 337 nM, 338 nM, 339 nM or 400 nMor at a concentration of about 339 nM.

In one variation cystine is present in the cell culture medium at aconcentration of from about 0.8 mM to about 2.5 mM or from about 0.8 mMto about 2.0 mM or from about 0.8 mM to about 1.75 mM or from about 0.8mM to about 1.5 mM or from about 1.0 mM to about 2.0 mM or from about1.0 mM to about 1.5 mM or from about 1.2 mM to about 1.4 mM or at aconcentration of about any one of 0.8 mM or 0.9 mM or 1.0 mM or 1.1 mMor 1.2 mM or 1.3 mM or 1.4 mM or 1.5 mM or at a concentration of aboutany one of 1.1 mM, 1.3 mM or 1.5 mM or at a concentration of about 1.3mM.

In one variation, an animal-derived hydrolysate is present in the cellculture media at a concentration of from about 5.6 g/L to about 38.0 g/Lor from about 7.0 g/L to about 35.0 g/L or from about 7.0 g/L to about25.0 g/L or from about 7.0 g/L to about 15.0 g/L or from about 8.0 g/Lto about 12.0 g/L or from about 7.0 g/L to about 11.0 g/L or about anyone of 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, 45g/L or 50 g/L or about any one of 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10g/L, 11 g/L, or 12 g/L or about 10 g/L.

In one variation, an plant-derived hydrolysate is present in the cellculture media at a concentration of from about 1.4 g/L to about 6.2 g/Lor from about 1.5 g/L to about 5.5 g/L or from about 1.5 g/L to about4.5 g/L or from about 1.5 g/L to about 3.5 g/L or from about 1.5 g/L toabout 2.5 g/L or from about 1.75 g/L to about 2.75 g/L or from about 2.0g/L to about 3.0 g/L or from about 2.25 g/L to about 2.75 g/L or aboutany one of 1.75 g/L, 2.0 g/L, 2.25 g/L, 2.5 g/L, 3.0 g/L, 3.25, 3.5 g/L,3.75 g/L, or 4.0 g/L or about any one of 2.0 g/L, 2.25 g/L, 2.5 g/L or3.0 g/L or about 2.5 g/L.

In a further variation, cysteine is present in the cell culture medium.Cysteine may in one aspect be added to a basal cell culture medium(e.g., by supplementing the basal cell culture medium with a feed mediumcomprising cysteine). In one variation, a cell culture medium comprisescysteine (which may be added to a basal cell culture medium that doesnot comprise cysteine via a feed medium comprising cysteine) in aconcentration of from about 0.5 mM to about 5.0 mM or from about 1.0 mMto about 12.0 mM or from about 2.0 mM to about 10.0 mM or from about 2.0mM to about 8.0 mM or about 1.0 mM to about 10.0 mM or from about 1.0 mMto about 8.0 mM or from about 2.0 mM to about 12.0 mM or from about 3.0mM to about 12.0 mM or from about 4.0 mM to about 12.0 mM or from about5.0 mM to about 12.0 mM or from about 6.0 mM to about 12.0 mM or fromabout 6.0 mM to about 10.0 mM or from about 6.0 mM to about 8.0 mM orabout any one of 0.5 mM, 0.8 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 5.0 mM,5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM or 9.0 mM. In oneaspect, cysteine is added to a basal cell culture media (which additionmay be at any point in time of the cell culture cycle and may be in oneor more amounts, which may be the same or different), in an amount suchthat cysteine is present in the cell culture media at a concentration ofabout 7.5 mM.

In a further variation, cystine is present in the cell culture medium.Cystine may in one aspect be added to a basal cell culture medium (e.g.,by supplementing the basal cell culture medium which may or may notalready comprise cystine with a feed medium comprising cystine). In onevariation, a cell culture medium comprises cystine (which may be addedto a basal cell culture medium via a feed medium comprising cystine) ina concentration of from about 0.5 mM to about 5.0 mM, such as in anamount to provide about 0.8 mM cysteine in the cell culture medium.

In certain embodiments, the cell culture medium comprises cystine but isfree of cysteine.

The cell culture medium (e.g., a basal cell culture medium) may furtherbe supplemented with an additional cell culture medium components (e.g.,such as via a feed cell culture medium). In one aspect, the additionalcell culture medium component comprises insulin. In another aspect, theadditional cell culture medium component comprises insulin and cysteine.A cell culture media provided herein may be supplemented with any amountof insulin and/or cysteine that is suitable for culturing a cell. In oneaspect, insulin is added to a cell culture medium (e.g., added to abasal cell culture medium at one or more point in time of the cellculture cycle) in an amount to provide a concentration of insulin in thecell culture of about 15 mg/L or about 25 mg/L. In one aspect, insulinis added to a cell culture medium (e.g., added to a basal cell culturemedium at one or more point in time of the cell culture cycle) in anamount to provide a concentration of insulin in the cell cultureselected from the group consisting of: from about 1.0 mg/L to about100.0 mg/L; from about 10.0 mg/L to about 100.0 mg/L; from about 10.0mg/L to about 50.0 mg/L; from about 10.0 mg/L to about 35.0 mg/L; fromabout 10.0 mg/L to about 25.0 mg/L; from about 5.0 mg/L to about 80.0mg/L; from about 5.0 mg/L to about 60.0 mg/L; from about 5.0 mg/L toabout 50.0 mg/L; from about 5.0 mg/L to about 30.0 mg/L; from about 5.0mg/L to about 25.0 mg/L; from about 10.0 mg/L to about 25.0 mg/L; fromabout 10.0 mg/L to about 30.0 mg/L; from about 15.0 mg/L to about 20.0mg/L; from about 5.0 mg/L to about 15.0 mg/L; from about 6.0 mg/L toabout 12.0 mg/L; from about 7.0 mg/L to about 11.0 mg/L; from about 8.0mg/L to about 10.0 mg/L; about any one of 5.0 mg/L, 6.0 mg/L, 7.0 mg/L,8.0 mg/L, 9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0 mg/L, 13.0 mg/L, 14.0mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0 mg/L, 19.0 mg/L, 20.0 mg/L,21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0 mg/L, 25.0 mg/L, 26.0 mg/L, 27.0mg/L, 28.0 mg/L, 29.0 mg/L or 30.0 mg/L; about any one of 7 mg/L, 8.0mg/L, 9.0 mg/L, 10.0 mg/L and 11.0 mg/L. In another aspect, cysteine isadded to a cell culture medium (e.g., added to a basal cell culturemedium at one or more point in time of the cell culture cycle) in anamount to provide a concentration of cysteine in the cell cultureselected from the group consisting of: from about 1.0 mM to about 12.0mM or from about 2.0 mM to about 10.0 mM or from about 2.0 mM to about8.0 mM or about 1.0 mM to about 10.0 mM or from about 1.0 mM to about8.0 mM or from about 2.0 mM to about 12.0 mM or from about 3.0 mM toabout 12.0 mM or from about 4.0 mM to about 12.0 mM or from about 5.0 mMto about 12.0 mM or from about 6.0 mM to about 12.0 mM or from about 6.0mM to about 10.0 mM or from about 6.0 mM to about 8.0 mM or about anyone of 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM or9.0 mM. In another aspect, cystine is added to a cell culture medium(e.g., added to a basal cell culture medium at one or more point in timeof the cell culture cycle) in an amount to provide a concentration ofcystine in the cell culture of from about 0.1 mM to about 1.5 mM, suchas a concentration of about 0.2 mM.

In some aspects, a cell culture medium provided herein comprises fromabout 5.0 mg/L to about 14.0 mg/L, from about 5.5 mg/L to about 13.0mg/L, from about 6.0 mg/L to about 12.0 mg/L, from about 7.0 mg/L toabout 11.0 mg/L, from about 8.0 mg/L to about 10.0 mg/L, or from about8.5 mg/L to about 14.0 mg/L insulin. It is understood that the cellculture medium comprising insulin may further comprise any one or moreof copper and cystine in any amount provided herein. For example, it isunderstood that a cell culture medium comprising from about 6.0 mg/L toabout 12.0 mg/L insulin may further comprise from about 70 nM to about400 nM of copper and/or from about 0.5 mM to about 2.5 mM cystine andmay further comprise an animal-derived hydrolysate and/or plant-derivedhydrolysate, for example an animal-derived hydrolysate from about 5.5g/L to about 40.0 g/L and/or a plant-derived hydrolysate from about 1.5g/L to about 6.5 g/L.

In other aspects, a cell culture medium provided herein comprises fromabout 65 nM to about 400 nM, from about 70 nM to about 375 nM, fromabout 75 nM to about 350 nM, from about 80 nM to about 325 nM, fromabout 85 nM to about 300 nM, or from about 90 nM to about 275 nM copper.It is understood that the cell culture medium comprising copper mayfurther comprise any one or more of insulin and cystine in any amountprovided herein. For example, it is understood that a cell culturemedium comprising from about 85 nM to about 300 nM copper may furthercomprise from about 0.8 mM to about 1.75 mM of cystine and/or from about8.0 mg/L to about 12.0 mg/L insulin and may further comprise ananimal-derived hydrolysate and/or plant-derived hydrolysate, for examplean animal-derived hydrolysate from about 5.5 g/L to about 40.0 g/Land/or a plant-derived hydrolysate from about 1.5 g/L to about 6.5 g/L.

In some aspects, a cell culture medium provided herein comprises fromabout 0.8 mM to about 1.75 mM, from about 0.9 mM to about 1.50 mM, fromabout 1.0 mM to about 1.40 mM, or from about 1.0 mM to about 1.30 mMcystine. It is understood that the cell culture medium comprisingcystine may further comprise any one or more of insulin and copper inany amount provided herein. For example, it is understood that a cellculture medium comprising from about 0.8 mM to about 1.75 mM cystine mayfurther comprise from about 70 nM to about 375 nM of copper and/or fromabout 8.0 mg/L to about 12.0 mg/L insulin and may further comprise ananimal-derived hydrolysate and/or plant-derived hydrolysate, for examplean animal-derived hydrolysate from about 5.5 g/L to about 40.0 g/Land/or a plant-derived hydrolysate from about 1.5 g/L to about 6.5 g/L.

In some embodiments, the cell culture medium further comprises ananimal-derived hydrolysate in amounts as described in Table 1. In otherembodiments, the cell culture medium further comprises a plant-derivedhydrolysate in amounts as described in Table 1. In some embodiments, thecell culture medium further comprises both an animal-derived hydrolysateand a plant-derived hydrolysate in amounts as described in Table 1.

In some aspects, a cell culture medium provided herein comprises fromabout 1.5 g/L to about 6.0 g/L, from about 2.0 g/L to about 5.5 g/L,from about 2.5 g/L to about 5.0 g/L, from about 3.0 g/L to about 4.5g/L, or from about 3.5 g/L to about 4.0 g/L plant-derived hydrolysate.It is understood that the cell culture medium comprising plant-derivedhydrolysate may further comprise any one or more of cystine, insulin andcopper in any amount provided herein. For example, it is understood thata cell culture medium comprising from about 0.8 mM to about 1.75 mMcystine may further comprise from about 70 nM to about 375 nM of copperand/or from about 8.0 mg/L to about 12.0 mg/L insulin and may furthercomprise an animal-derived hydrolysate, for example an animal-derivedhydrolysate from about 6.0 g/L to about 20.0 g/L.

In some aspects, a cell culture medium provided herein comprises fromabout 6.0 g/L to about 35.0 g/L, from about 7.0 g/L to about 30.0 g/L,from about 8.0 g/L to about 25.0 g/L, from about 9.0 g/L to about 20g/L, or from about 10.0 g/L to about 15.0 g/L animal-derivedhydrolysate. It is understood that the cell culture medium comprisinganimal-derived hydrolysate may further comprise any one or more ofcystine, insulin and copper in any amount provided herein. For example,it is understood that a cell culture medium comprising from about 0.8 mMto about 1.75 mM cystine may further comprise from about 70 nM to about375 nM of copper and/or from about 8.0 mg/L to about 12.0 mg/L insulinand may further comprise an plant-derived hydrolysate, for example anplant-derived hydrolysate from about 1.5 g/L to about 3.0 g/L.

In some of the embodiments herein, the cell culture medium comprisesfrom about 0.9 mM to about 1.5 mM cystine. In some of the embodimentsherein, the cell culture medium comprises from about 1.4 mg/L to about11.0 mg/L insulin or from about 1.44 mg/L to about 66.0 mg/L insulin. Insome of the embodiments herein, the cell culture medium comprises fromabout 26.0 nM to about 400.0 nM copper. In some aspects, a cell culturemedium comprises two or more components selected from: (a) about 69.0 nMto about 400.0 nM copper, (b) from about 7.0 mg/L to about 11.0 mg/Linsulin or from about 1.44 mg/L to about 66 mg/L insulin, and (c) fromabout 0.8 mM to about 2.5 mM cystine.

Cell culture media components described herein (e.g., a cell culturemedia comprising any one or more of copper, insulin, cystine, ananimal-derived hydrolysate and a plant-derived hydrolysate) may be addedto a cell culture medium in a form that is known in the art, such as asalt, a hydrate or combination thereof. The cell culture mediacomponents can also be provided to the cell culture media as a solution,an extract, or in solid form. In some embodiments, the copper isprovided to the cell culture medium as CuSO4. As a non-limiting example,cystine may be provided to the cell culture medium as the disodium saltmonohydrate powder. Protein hydrolysates, also known as peptones, aretypically manufactured by enzymatic digestion of a variety ofbiologically based starting materials such as animal tissues,milk-derived products, microorganisms or plants. The hydrolysate used inthe cell culture medium provided herein can be derived from a plant oran animal (e.g., plant-derived hydrolysate and/or animal-derivedhydrolysate). A plant hydrolysate as described herein can be derivedfrom, but not limited to, wheat gluten, maize, cereal, soy, orcottonseed. An animal hydrolysate as described herein can be derivedfrom, but not limited to, bovine, chicken, caprine, equine, human,ovine, porcine, or rabbit or other animals.

A method of preparing a cell culture medium for use in culturing amammalian cell comprising a nucleic acid encoding bevacizumab, or afragment thereof, is also provided, wherein the method comprisescombining any two or more of copper, insulin and cystine in acomposition suitable for cell culture. In one aspect, the methodcomprises adding any two or more of copper, insulin and cystine to acomposition suitable for cell culture, wherein the two or more ofcopper, insulin and cystine may be added to the composition sequentiallyor simultaneously. In a further variation, a method of preparing a cellculture medium for use in culturing a mammalian cell comprising anucleic acid encoding bevacizumab, or a fragment thereof, is provided,wherein the method comprises combining any two or more of copper,insulin and cystine in a composition suitable for cell culture at afirst period of time and wherein the method further comprises adding anamount of insulin at a second period of time, such as at least once, atleast twice, at least three times, at least four time, at least fivetimes, at least six times, at least seven times, etc. of a cell growthcycle. In some embodiments, a cell growth cycle is at least 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18, days,19 days, 20 days, or any amount of days wherein the cells may remain incell culture while still remaining viable. In one variation of a methodof preparing a cell culture medium, cystine is added in an amount toprovide about 0.9 mM to about 1.5 mM cystine in the cell culture medium.In another variation of a method of preparing a cell culture medium,insulin is added in an amount to provide from about 1.4 mg/L to about11.0 mg/L insulin or from about 1.44 mg/L to about 66.0 mg/L insulin inthe cell culture medium. In another variation of a method of preparing acell culture medium, copper is added in an amount to provide from about26.0 nM to about 400.0 nM copper in the cell culture medium. In someaspects, a cell culture medium is prepared by combining two or morecomponents selected from: (a) copper in an amount to provide about 69.0nM to about 400.0 nM copper in the cell culture medium, (b) insulin inan amount to provide from about 7.0 mg/L to about 11.0 mg/L or fromabout 1.44 mg/L to about 66 mg/L insulin in the cell culture medium, and(c) cystine in an amount to provide from about 0.8 mM to about 2.5 mMcystine in the cell culture medium.

In some embodiments herein, the cell culture medium is a basal cellculture medium. In other embodiments herein, the cell culture medium isa feed cell culture medium. In some embodiments herein, the cell culturemedium is a basal cell culture medium comprising at least one of copper,insulin, and cystine, and where the basal cell culture medium issupplemented (e.g., at a period of time following initiation of a cellculture cycle, such as any one of at least two times, at least threetimes, at least four times, at least five times, at least six times, atleast seven times, etc. of a cell culture cycle) with a feed cellculture medium comprising any one or more of insulin, an animal-derivedhydrolysate and a plant-derived hydrolysate.

Individual media components provided herein may be present in amountsthat result in one or more advantageous properties for culturing cellscomprising a nucleic acid encoding bevacizumab, or a fragment thereof,and/or for bevacizumab production from cell culture. Advantageousproperties include, but are not limited to, increased cell viability,increase in the amount of bevacizumab produced from the cell (e.g.,enhanced bevacizumab titer) and/or reduced oxidation of bevacizumab incell culture. Advantageous properties of the cell culture media providedherein may also include maintaining or enhancing the amount ofbevacizumab produced by the cells (e.g., antibody titer) whilemaintaining the N-glycosylation profile, the charge heterogeneity and/orthe amino acid sequence integrity of bevacizumab, or a fragment thereof.These advantageous properties are applicable to methods of culturing acell comprising a nucleic acid encoding bevacizumab, or a fragmentthereof and to methods of producing bevacizumab, or a fragment thereofin cell culture as described herein.

A cell culture medium provided herein in one aspect results in one ormore favorable product quality attribute or advantageous property whenused in a method of producing bevacizumab, or a fragment thereof. In onevariation, use of the cell culture medium provided herein increases theamount of bevacizumab produced by the cells (e.g., enhances antibodytiter) as compared to the amount of bevacizumab, or a fragment thereof,produced by culturing the cell producing bevacizumab in a different cellculture medium.

As would be understood by the skilled artisan, the cell culture mediadetailed herein may comprise other components (e.g., besides the one ormore of copper, insulin, and cystine, and optionally peptonehydrolysate) that are useful for cell culture. For example, it isunderstood that the cell culture media may comprise additionalcomponents such as amino acids (e.g., glutamine, arginine, orasparagine), vitamins (including but not limited to B vitamins such asany one or more of vitamin B1, vitamin B2, vitamin B3, vitamin B6,vitamin B7, vitamin B9, or vitamin B12), trace elements, transitionmetals (including but not limited to nickel, iron (e.g., ferric iron orferrous iron), or zinc), and other media components. Any media providedherein may also be supplemented with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor), ions(such as sodium, chloride, calcium, magnesium, and phosphate), buffers(such as HEPES), nucleosides (such as adenosine and thymidine), traceelements and glucose or an equivalent energy source. Additional cellculture media components, such as those listed herein, may be includedin the cell culture medium at appropriate concentrations that would beknown to those skilled in the art.

III. Methods and Uses of the Invention

Provided herein are methods of culturing cells used in the productionbevacizumab, or a fragment thereof, and use of cell culture media thatcomprise one or more of copper, insulin and cystine. In some aspects, amethod is provided for culturing a mammalian cell comprising a nucleicacid encoding bevacizumab or fragment thereof, wherein the methodcomprises the step of contacting the mammalian cell with a cell culturemedium comprising at least two of copper, insulin and cystine, whereinthe cell culture medium may additionally comprise a plant-derivedhydrolysate, an animal-plant-derived hydrolysate or both a plant-derivedhydrolysate and an animal-derived hydrolysate. In some embodiments, thecell culture media comprises insulin. In some of the embodiments herein,the cell culture media comprises copper. In some of the embodimentsherein, the cell culture media comprises cystine. In some of theembodiments herein, the cell culture media comprises copper and cystine.In some of the embodiments herein, the cell culture media comprisescopper and insulin. In some of the embodiments herein, the cell culturemedia comprises insulin and cystine. In some of the embodiments herein,the amount of the components in the cell culture medium (e.g., theamount of copper, insulin, cystine, plant-derived hydrolysate and/oranimal-derived hydrolysate) is in an amount selected from a valueprovided in Table 1. In some embodiments, the method further comprisesthe step of adding an additional amount of insulin to the medium. Theadditional amount of insulin can be added to the cell culture medium atleast once, at least three times, at least 6 times or at least 12 timesduring the cell culture cycle. In some of the embodiments herein, theadditional amount of insulin added to the cell culture is added in anamount to provide insulin in the cell culture medium at a concentrationselected from Table 1 such as from about 1 mg/L to about 44 mg/L. Insome aspects, the cell culture medium further comprises ananimal-derived hydrolysate, a plant-derived hydrolysate, or both ananimal-derived hydrolysate and a plant-derived hydrolysate.

In some other aspects, a method is provided for culturing a cellcomprising a nucleic acid encoding bevacizumab or fragment thereof,wherein the method comprises the step of contacting the cell with a cellculture medium comprising two or more components selected from the groupconsisting of copper, insulin, and cystine. In some embodiments herein,the cell culture medium comprises insulin at a concentration of fromabout 7.0 mg/L to about 11.0 mg/L. In some embodiments herein, the cellculture medium comprises copper at a concentration of from about 69.0 nMto about 400.0 nM. In some embodiments herein, the cell culture mediumcomprises cystine at a concentration of from about 0.8 mM to about 2.5mM. In some embodiments herein the cell culture medium comprises insulinat a concentration of from about 7.0 mg/L to about 11.0 mg/L and copperat a concentration of from about 69.0 nM to about 400.0 nM. In someembodiments herein the cell culture medium comprises insulin at aconcentration of from about 7.0 mg/L to about 11.0 mg/L and cystine at aconcentration of from about 0.8 mM to about 2.5 mM. In some embodimentsherein the cell culture medium comprises copper at a concentration offrom about 69.0 nM to about 400.0 nM and cystine at a concentration offrom about 0.8 mM to about 2.5 mM. In some embodiments herein the cellculture medium comprises insulin at a concentration of from about 7.0mg/L to about 11.0 mg/L, copper at a concentration of from about 69.0 nMto about 400.0 nM and cystine at a concentration of from about 0.8 mM toabout 2.5 mM. In any of the embodiments herein, the cell culture mediummay comprise cystine, insulin and/or copper in an amount selected fromTable 1. In some of the embodiments herein, the method further comprisesthe step of adding an additional amount of insulin to the cell culturemedium provided herein (e.g., such as via a feed medium introduced tothe basal cell culture medium at a period of time following initiationof the cell culture cycle). The additional amount of insulin can beadded to the cell culture medium at least once, at least two times, atthree times, at least six times, at least nine times, at least twelvetimes, or at least fourteen times during the cell culture cycle. In someof the embodiments herein, the additional amount of insulin added to thecell culture is added in an amount to provide insulin in the cellculture medium at a concentration selected from Table 1 such as 5.6 mg/Lto about 66 mg/L. In some aspects, the cell culture medium furthercomprises an animal-derived hydrolysate, a plant-derived hydrolysate, orboth an animal-derived hydrolysate and a plant-derived hydrolysate. Insome of the embodiments herein, the method further comprises the step ofadding an additional amount of animal-derived hydrolysate andplant-derived hydrolysate to the cell culture medium provided herein(e.g., such as via a feed medium introduced to the basal cell culturemedium at a period of time following initiation of the cell culturecycle). In some of the embodiments herein, the additional amount ofanimal-derived hydrolysate and plant-derived hydrolysate added to thecell culture is added in an amount to provide animal-derived hydrolysateand plant-derived hydrolysate in the cell culture medium at aconcentration selected from the concentrations listed in Table 1.

In another aspect, provided herein are methods of producing bevacizumabor a fragment thereof, wherein the method comprises the step ofcontacting a cell capable of producing bevacizumab or a fragment thereofwith a cell culture medium comprising two or more components selectedfrom the group consisting of copper, insulin, and cystine. In someembodiments herein, the cell culture medium comprises insulin at aconcentration of from about 7.0 mg/L to about 11.0 mg/L. In someembodiments herein, the cell culture medium comprises copper at aconcentration of from about 69.0 nM to about 400.0 nM. In someembodiments herein, the cell culture medium comprises cystine at aconcentration of from about 0.8 mM to about 2.5 mM. In any of theembodiments herein, the cell culture medium may comprise cystine,insulin or copper in an amount selected from Table 1. In some of theembodiments herein, the method further comprises the step of adding anadditional amount of insulin to the cull culture medium provided herein.Insulin may be added to the cell culture medium in any amount that issuitable for cell culture. In one aspect, insulin is added to the cellculture medium in an amount to provide insulin in the cell culturemedium at a concentration selected from the concentrations listed inTable 1. In a particular aspect, insulin is added to the cell culturemedium in an amount to provide insulin in the cell culture medium at aconcentration selected from the group consisting of: from about 1.0 mg/Lto about 100.0 mg/L; from about 5.0 mg/L to about 80.0 mg/L; from about5.0 mg/L to about 60.0 mg/L; from about 5.0 mg/L to about 50.0 mg/L;from about 5.0 mg/L to about 40.0 mg/L; from about 5.0 mg/L to about30.0 mg/L; from about 5.0 mg/L to about 25.0 mg/L; from about 10.0 mg/Lto about 25.0 mg/L; from about 10.0 mg/L to about 30.0 mg/L; from about15.0 mg/L to about 20.0 mg/L; from about 5.0 mg/L to about 15.0 mg/L;from about 6.0 mg/L to about 12.0 mg/L; from about 7.0 mg/L to about11.0 mg/L and from about 8.0 mg/L to about 10.0 mg/L. In another aspect,insulin is added to the cell culture medium in an amount to provideinsulin in the cell culture medium at a concentration of about any oneof 5.0 mg/L, 6.0 mg/L, 7.0 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L, 11.0mg/L, 12.0 mg/L, 13.0 mg/L, 14.0 mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L,18.0 mg/L, 19.0 mg/L, 20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0mg/L, 25.0 mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0mg/L. In a further aspect, insulin is added to the cell culture mediumin an amount to provide insulin in the cell culture medium at aconcentration of about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/Lor 11.0 mg/L. The additional amount of insulin can be added to the cellculture medium at any time during the cell culture cycle. For example,insulin may be added at any one or more of days 1-20 for a 20 day cellculture cycle (e.g., at any one or more of days 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). When an additionalamount of insulin is added, it may be added in any amount, which amountmay be the same or different when insulin is added more than once duringa cell culture cycle. It is therefore appreciated that for a 14 day cellculture cycle, insulin may be added at any one or more of days 1-14(e.g., at any one or more of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14) in any amount, which amount may be the same or different wheninsulin is added more than one during a cell culture cycle. Theadditional amount of insulin can be added to the cell culture medium atleast once, at least two times, at three times, at least six times, atleast nine times, at least twelve times, or at least fourteen timesduring the cell culture cycle. In some of the embodiments herein, theadditional amount of insulin added to the cell culture is added in anamount to provide insulin in the cell culture medium at a concentrationselected from Table 1 such as 5.6 mg/L to about 66 mg/L. In someaspects, the cell culture medium further comprises an animal-derivedhydrolysate, a plant-derived hydrolysate, or both an animal-derivedhydrolysate and a plant-derived hydrolysate. In some of the embodimentsherein, the method further comprises the step of adding an additionalamount of animal-derived hydrolysate and plant-derived hydrolysate tothe cell culture medium provided herein (e.g., such as via a feed mediumintroduced to the basal cell culture medium at a period of timefollowing initiation of the cell culture cycle). In some of theembodiments herein, the additional amount of animal-derived hydrolysateand plant-derived hydrolysate added to the cell culture is added in anamount to provide animal-derived hydrolysate and plant-derivedhydrolysate in the cell culture medium at a concentration selected fromthe concentrations listed in Table 1.

In another aspect, provided herein are methods of producing bevacizumabor a fragment thereof, comprising a step of culturing a mammalian cellcomprising a nucleic acid encoding bevacizumab or a fragment thereof ina cell culture medium, wherein initial cell culture medium in a cellculture cycle comprises two or more components selected from the groupconsisting of copper at a concentration of from about 69 nM to about1,000 nM, insulin at a concentration of from about 1.0 mg/L to about100.0 mg/L, and cystine at a concentration of from about 0.7 mM to about2.0 mM, and wherein the cell produces bevacizumab or the fragment. Insome embodiments, the initial cell culture medium comprising (1) copperand insulin; (2) copper and cystine; (3) insulin and cystine; or (4)copper, insulin, and cystine. In some embodiments, the initial cellculture medium comprises copper, cystine, and/or insulin in aconcentration selected from the concentrations as described in Table 1.In some embodiments, the initial cell culture medium comprises ananimal-derived hydrolysate and/or a plant-derived hydrolysate in aconcentration selected from the concentrations as described in Table 1.In some embodiments, the initial cell culture medium comprises insulinand the method further comprises a step of adding an additional amountof insulin to the cell culture medium during the cell culture cycle. Insome embodiments, the additional amount of insulin is added to the cellculture medium at least once, at least twice, at least three times, atleast four times, at least five times, or at least six times during thecell culture cycle. In some embodiments, the insulin added each time isfrom about 5 mg/L to about 25 mg/L (e.g., about any one of 5 mg/L, 10mg/L, 15 mg/L, 20 mg/L and 25 mg/L). In some embodiments, the cumulativeamount of insulin added during the cell culture cycle is from about 20mg/L to about 100 mg/L (e.g., about any one of 20 mg/L, 25 mg/L, 30mg/L, 35 mg/L, 40 mg/L, 45 mg/L, 50 mg/L, 55 mg/L, 60 mg/L, 65 mg/L, 70mg/L, 75 mg/L, 80 mg/L, 85 mg/L, 90 mg/L, 95 mg/L, and 100 mg/L). Insome embodiments, the initial cell culture medium comprises cystine andthe method further comprises a step of adding an additional amount ofcystine to the cell culture medium during the cell culture cycle. Forexample, cystine is added in an amount to provide from about 0.1 toabout 1.5 mM additional cystine in the cell culture medium (e.g., about0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about0.6 mM, about 0.7 mM, about 1 mM, or about 1.5 mM additional cystine inthe cell culture medium). In some embodiments, cystine is added in abatch feed during the cell culture cycle. In some embodiments, themethod further comprises at least one batch feed during the cell culturecycle (e.g., two, three, or four batch feeds during the cell culturecycle). In some embodiments, the batch feed medium further comprises ananimal-derived hydrolysate and/or a plant-derived hydrolysate (e.g., ina concentration selected from the concentrations as described in Table1). In some embodiments, during the cell culture cycle, the temperatureof the medium is reduced by at least about 2, at least about 3, at leastabout 4, or at least about 5 degrees C. relative to the temperature atthe beginning of the culturing. In some embodiments, the temperature ofthe medium is reduced at least once or at least twice during the cellculture cycle. In some embodiments, the temperature is reduced on day 8and day 10 after the beginning of the culturing. In some embodiments,the cell is cultured at a temperature ranging from about 31° C. to about35° C. In some embodiments, the cell is cultured at a first temperatureof about 35° C. for a first period of time, is cultured at a secondtemperature of about 33° C. for a second period of time, and is culturedat a third temperature of about 31° C. for a third period of time. Insome embodiments, the cell is cultured in the medium having a pH atabout 7.0 to about 7.3.

As used herein, the term “initial cell culture medium” refers to thecell culture medium at the beginning of a cell culture cycle. In someembodiments, the initial cell culture medium is the cell culture mediumafter cells are inoculated in a basal medium.

As used herein, the term “cumulative” refers to the total amount of aparticular component or components added over the cell culture cycle,including components added at the beginning of the cell culture cycleand subsequently added components. In some embodiments, the cumulativeamount is the total concentration of a component or components addedinto the cell culture medium as measured or calculated in the cellculture medium after addition. In some embodiments, the component orcomponents are added into the cell culture medium by a feed solutioncontaining concentrated component or components. For example, if theinitial culture medium has 10 mg/L insulin, and additional amount ofinsulin at 15 mg/L is added during the cell culture cycle to achieve anincrease of 15 mg/L in the cell culture medium, the cumulative amount ofthe insulin added during the cell culture cycle is 25 mg/L.

In some embodiments, a cell culture cycle refers to a period of timefrom inoculating cells into a basal cell culture medium for producing apolypeptide expressed by the cells to the end of the production period.In some embodiments, the cell culture cycle is at least 10 days, atleast 11 days, at least 12 days, at least 13 days, at least 14 days, atleast 15 days, at least 16 days, at least 17 days, at least 18 days, atleast 19 days, at least 20 days, at least 21 days, at least 22 days, atleast 23 days, or any amount of days wherein the cells may remain viablefor producing the polypeptide expressed by the cells.

In some of the embodiments herein, the method increases the amount ofbevacizumab or fragment thereof produced by the mammalian cell ascompared to a the amount of bevacizumab or fragment thereof themammalian cell produces when cultured in a cell culture medium that doesnot comprise one or more of components listed in Table 1. In someembodiments, the amount of bevacizumab or fragment thereof produced by acell cultured in a cell culture medium comprising at least two ofcopper, insulin and cystine is increased by at least 5%, 6,%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, or 50% as compared to the amount of bevacizumab or fragmentthereof produced by the cell when cultured in a cell culture medium thatdoes not comprise at least two of copper, insulin and cystine.

The cell culture medium provided herein can be used a basal cell culturemedium and/or as a feed cell culture medium. In some embodiments, a cellculture medium provided herein is used in a method for culturing thecell during the cell's growth phase. In some embodiments, a cell culturemedium provided herein is used in a method for culturing the cell duringthe cell's production phase.

It is understood that any of the methods detailed herein including: (i)a method of producing bevacizumab, or a fragment thereof; (ii) a methodof culturing a mammalian cell comprising a nucleic acid encodingbevacizumab, or a fragment thereof; and (iii) a method of enhancingproduction of bevacizumab, or a fragment thereof, (e.g., enhancing titeryields of bevacizumab, or a fragment thereof) from a mammalian cellcomprising a nucleic acid encoding bevacizumab, or a fragment thereof,may be carried out in any suitable scale (e.g., any scale that producesbevacizumab, or a fragment thereof). In one aspect, any of the methodsdetailed herein are performed on a scale that is commensurate in scopewith commercial production of bevacizumab, or a fragment thereof. Forexample, in one variation, a cell capable of producing bevacizumab maybe cultured in a cell culture medium provided herein wherein theculturing occurs in a culturing vessel that is capable of holding acommercial batch of bevacizumab, such as in a culturing vessel capableof holding at least 10,000 L of cell culture (e.g., the methods in oneaspect are carried out on at least a 10,000 L cell culture scale, suchas a 12,000 L cell culture scale).

In further embodiments of the methods provided herein, the bevacizumabor a fragment thereof is recovered from the cell culture. A compositioncomprising the recovered bevacizumab or a fragment thereof can besubjected to at least one purification step before assessment of, e.g.,a quality attribute. In a further embodiment, the composition is apharmaceutical composition comprising bevacizumab or a fragment thereofand a pharmaceutically acceptable carrier.

Other methods and cell culture media are provided throughout, such as inthe Brief Summary of the Invention and elsewhere.

Polypeptide Production

The cell culture media detailed herein can be used in a method ofculturing cells to produce bevacizumab or a fragment thereof. The mediummay be used in a method of culturing cells capable of producingbevacizumab or a fragment thereof, whether by batch culture, fed batchculture or perfusion culture. In one embodiment, bevacizumab or afragment thereof is directly secreted into the medium by the host cell.In another embodiment, bevacizumab or a fragment thereof is releasedinto the medium by lysis of a cell comprising a nucleic acid encodingthe antibody or fragment thereof.

Bevacizumab or a fragment thereof that is expressible in a host cell maybe produced in accordance with the present disclosure and may be presentin the compositions provided.

Methods for producing antibodies and fragments thereof, in cell cultureare well known in the art. Provided herein are non-limiting exemplarymethods for producing an antibody (e.g., full length antibodies,antibody fragments and multispecific antibodies) in cell culture. SeeMolecular Cloning: A Laboratory Manual (Sambrook et al., 4^(th) ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012);Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,2003); Short Protocols in Molecular Biology (Ausubel et al., eds., J.Wiley and Sons, 2002); Current Protocols in Protein Science, (Horswillet al., 2006); Antibodies, A Laboratory Manual (Harlow and Lane, eds.,1988); Culture of Animal Cells: A Manual of Basic Technique andSpecialized Applications (R. I. Freshney, 6^(th) ed., J. Wiley and Sons,2010) for generally well understood and commonly employed techniques andprocedures for the production of antibodies (e.g., bevacizumab), whichare all incorporated herein by reference in their entirety.

Cell Culture and Antibody Production

Generally the cells are combined (contacted) with any of the cellculture media described herein under one or more conditions that promoteany of cell growth, maintenance and/or antibody production. Methods ofculturing a cell and producing an antibody employ a culturing vessel(bioreactor) to contain the cell and cell culture medium. The culturingvessel can be composed of any material that is suitable for culturingcells, including glass, plastic or metal. Typically, the culturingvessel will be at least 1 liter and may be 10, 100, 250, 500, 1000,2500, 5000, 8000, 10,000 liters or more (e.g., a 12,000 liter vessel).In one aspect the culturing vessel is capable of containing at least 2liters, at least 10 liters, at least 100 liters, at least 500 liters, atleast 1,000 liters, at least 2,500 liters, at least 5,000 liters, atleast 7,500 liters, at least 10,000 liters, at least 12,000 liters ormore of a cell culture medium provided herein as is required forproducing manufacturing scale amounts of bevacizumab from cell culture.Thus, the compositions and methods provided herein may find use in amanufacturing-scale production of bevacizumab, or a fragment thereof.The culture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan. Culturing conditions thatmay be adjusted during the culturing process include but are not limitedto pH and temperature. In some of the embodiments herein, the pH is atleast 7.0, 7.15, 7.2, 7.25, 7.30, 7.35, 7.4, 7.45, or 7.50 but no morethan 8.0. The number of cells comprising a nucleic acid encodingbevacizumab, or a fragment thereof, that can be inoculated into a cellculture medium provided herein will be apparent to one of skill in theart. For example, about 1.0×10⁶ to about 2.0×10⁶ cells (including any ofabout 1.1×10⁶, about 1.2×10⁶ about 1.3×10⁶, about 1.4×10⁶, about1.5×10⁶, about 1.6×10⁶, about 1.7×10⁶, about 1.8×10⁶ or about 1.9×10⁶)comprising a nucleic acid encoding bevacizumab, or a fragment thereof,can be inoculated in a medium provided herein for initiation of a cellculture cycle. In one aspect, the number of cells comprising a nucleicacid encoding bevacizumab, or a fragment thereof, that can be inoculatedinto a cell culture medium provided herein is from about 1.2×10⁶ toabout 1.8×10⁶ cells or from about 1.3×10⁶ to about 1.7×10⁶ cells or fromabout 1.5×10⁶ to about 1.7×10⁶ cells.

A cell culture is generally maintained in the initial growth phase underconditions conducive to the survival, growth and viability (maintenance)of the cell culture. The precise conditions will vary depending on thecell type, the organism from which the cell was derived, and the natureand character of the expressed antibody or fragment thereof.

The temperature of the cell culture in the initial growth phase will beselected based primarily on the range of temperatures at which the cellculture remains viable. For example, during the initial growth phase,CHO cells grow well at 37° C. In general, most mammalian cells grow wellwithin a range of about 25° C. to 42° C. Preferably, mammalian cellsgrow well within the range of about 35° C. to 40° C. Those of ordinaryskill in the art will be able to select appropriate temperature ortemperatures in which to grow cells, depending on the needs of the cellsand the production requirements.

In one embodiment of the present invention, the temperature of theinitial growth phase is maintained at a single, constant temperature. Inanother embodiment, the temperature of the initial growth phase ismaintained within a range of temperatures. For example, the temperaturemay be steadily increased or decreased during the initial growth phase.Alternatively, the temperature may be increased or decreased by discreteamounts at various times during the initial growth phase. One ofordinary skill in the art will be able to determine whether a single ormultiple temperatures should be used, and whether the temperature shouldbe adjusted steadily or by discrete amounts.

The cells may be cultured during the initial growth phase for a greateror lesser amount of time. In one variation, the cells are cultured for aperiod of time sufficient to achieve a viable cell density that is agiven percentage of the maximal viable cell density that the cells wouldeventually reach if allowed to grow undisturbed. For example, the cellsmay be cultured for a period of time sufficient to achieve a desiredviable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 99 percent of maximal viable cell density.

In another embodiment the cells are allowed to grow for a defined periodof time. For example, depending on the starting concentration of thecell culture, the temperature at which the cells are cultured, and theintrinsic growth rate of the cells, the cells may be cultured for 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore days. In some cases, the cells may be allowed to grow for a monthor more.

The cell culture may be agitated or shaken during the initial culturephase in order to increase oxygenation and dispersion of nutrients tothe cells. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor during theinitial growth phase, including but not limited to pH, temperature,oxygenation, etc. For example, pH can be controlled by supplying anappropriate amount of acid or base and oxygenation can be controlledwith sparging devices that are well known in the art.

An initial culturing step is a growth phase, wherein batch cell cultureconditions are modified to enhance growth of recombinant cells, toproduce a seed train. The growth phase generally refers to the period ofexponential growth where cells are generally rapidly dividing, e.g.growing. During this phase, cells are cultured for a period of time,usually 1 to 4 days, e.g. 1, 2, 3, or 4 days, and under such conditionsthat cell growth is optimal. The determination of the growth cycle forthe host cell can be determined for the particular host cell by methodsknown to those skilled in the art.

In the growth phase, a basal culture medium provided herein and cellsmay be supplied to the culturing vessel in batch. The culture medium inone aspect contains less than about 5% or less than 1% or less than 0.1%serum and other proteins derived from plants or animals (e.g.,animal-derived hydrolysates and/or plant-derived hydrolysates). In someembodiments, the basal medium does not comprise an animal-derived orplant-derived hydrolysate. However, serum and animal-derived proteinscan be used if desired. At a particular point in their growth, the cellsmay form an inoculum to inoculate a culture medium at the start ofculturing in the production phase. Alternatively, the production phasemay be continuous with the growth phase. The cell growth phase isgenerally followed by a polypeptide production phase (e.g., antibodyproduction phase).

During the polypeptide production phase, the cell culture may bemaintained under a second set of culture conditions (as compared to thegrowth phase) conducive to the survival and viability of the cellculture and appropriate for expression of the desired polypeptide (e.g.,bevacizumab or fragment thereof). For example, during the subsequentproduction phase, CHO cells express recombinant polypeptides well withina range of 25° C. to 35° C. Multiple discrete temperature shifts may beemployed to increase cell density or viability or to increase expressionof the recombinant polypeptide. In one embodiment, a method ofincreasing polypeptide production (e.g., increasing production ofbevacizumab or fragment thereof) comprises a one or more temperatureshift step during the polypeptide production phase. In a furtherembodiment, a one or more temperature shift step comprises a shift ofthe temperature from 37° C. to 35° C., from 35° C. to 333° C., or from33° C. to 31° C. In some embodiments herein, a one or more temperatureshift step comprises a shift of temperature from about 37° C. on day 0to 35° C. on day 1 to 33° C. on day 8 and to 31° C. on day 10. In someembodiments herein, a one or more temperature shift step comprises ashift of temperature from about 37° C. on day 0 to 35° C. on day 3 to33° C. on day 8 and to 31° C. on day 10. In some embodiments herein, aone or more temperature shift step comprises a shift of temperature fromabout 37° C. on day 0 to 34° C. on day 2.5.

The cells may be maintained in the subsequent production phase until adesired cell density or production titer is reached. In one embodiment,the cells are maintained in the subsequent production phase until thetiter of the recombinant polypeptide (e.g., bevacizumab or fragmentthereof) reaches a maximum. In other embodiments, the culture may beharvested prior to this point. For example, the cells may be maintainedfor a period of time sufficient to achieve a viable cell density of 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 99 percent of maximal viable cell density. In some cases, it maybe desirable to allow the viable cell density to reach a maximum, andthen allow the viable cell density to decline to some level beforeharvesting the culture.

In certain cases, it may be beneficial or necessary to supplement thecell culture during the subsequent production phase with nutrients orother medium components that have been depleted or metabolized by thecells. For example, it might be advantageous to supplement the cellculture with nutrients or other medium components observed to have beendepleted during monitoring of the cell culture. Alternatively oradditionally, it may be beneficial or necessary to supplement the cellculture prior to the subsequent production phase. As non-limitingexamples, it may be beneficial or necessary to supplement the cellculture with hormones and/or other growth factors, particular ions (suchas sodium, chloride, calcium, magnesium, and phosphate), buffers,vitamins, nucleosides or nucleotides, trace elements (inorganiccompounds usually present at very low final concentrations), aminoacids, lipids, or glucose or other energy source. In one aspect, a basalcell culture is supplemented with insulin and/or plant-derivedhydrolysate and/or an animal-derived hydrolysate as detailed herein.

In some embodiments herein, the methods of the invention comprise thesupplementation of an additional amount of insulin into the cell cultureduring the cell production phase. For example, an additional 15 mg/L ofinsulin may be added to the cell culture on day of the production phaseof the cell culture cycle. In another example, an additional 5 mg/L ofinsulin may be added to the cell culture at least three times during theproduction phase of the cell culture cycle. In still another example, anadditional 5 mg/L of insulin may be added to the cell culture at leastsix times during the production phase of the cell culture cycle. A cellculture cycle may be at least 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days long. Insome embodiments, the cell culture cycle is up to 20 days long. In someembodiments, the cell culture is at least 20 days long. In someembodiments, a cell may be cultured for more than one cell culturecycle. In some of the embodiments herein, the method further comprisesthe step of adding an additional amount of animal-derived hydrolysateand plant-derived hydrolysate to the cell culture medium provided herein(e.g., such as via a feed medium introduced to the basal cell culturemedium at a period of time following initiation of the cell culturecycle). In some of the embodiments herein, the additional amount ofanimal-derived hydrolysate and plant-derived hydrolysate added to thecell culture is added in an amount to provide animal-derived hydrolysateand plant-derived hydrolysate in the cell culture medium at aconcentration selected from the concentrations listed in Table 1.

Antibody Purification

Bevacizumab or a fragment thereof preferably is recovered from theculture medium as a secreted polypeptide, although it also may berecovered from host cell lysates when directly expressed without asecretory signal.

The culture medium or lysate may be centrifuged to remove particulatecell debris. Bevacizumab or a fragment thereof thereafter may bepurified from contaminant soluble proteins and polypeptides, with thefollowing procedures being exemplary of suitable purificationprocedures: by fractionation on immunoaffinity or ion-exchange columns;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; and protein A Sepharose columns to remove contaminantssuch as IgG. A protease inhibitor such as phenyl methyl sulfonylfluoride (PMSF) also may be useful to inhibit proteolytic degradationduring purification. One skilled in the art will appreciate thatpurification methods suitable for the antibody or fragment thereof ofinterest may require modification to account for changes in thecharacter of the antibody or fragment thereof upon expression inrecombinant cell culture. An antibody or fragment thereof can begenerally purified using chromatographic techniques (e.g., affinitychromatography with a low pH elution step and ion exchangechromatography to remove process impurities). Purified bevacizumab or afragment thereof may be concentrated to provide a concentrated proteincomposition, e.g., one with an antibody concentration of at least 100mg/mL or 125 mg/mL or 150 mg/mL or a concentration of about 100 mg/mL or125 mg/mL or 150 mg/mL. It is understood that concentrated polypeptideproducts may be concentrated up to levels that are permissible under theconcentration conditions, e.g., up to a concentration at which thepolypeptide is no longer soluble in solution. Non-limiting examples ofmethods for producing and purifying antibodies for drug formulations aredescribed in Kelley, B. MAbs., 2009, 1(5):443-452, which is incorporatedherein in its entirety by reference.

IV. Pharmaceutical Formulations

Compositions comprising the cell culture medium provided herein and oneor more other component, such as a cell or a desired antibody orfragment thereof (i.e., bevacizumab or fragment thereof), are alsoprovided. A mammalian cell comprising a nucleic acid encodingbevacizumab or fragment thereof can secrete the antibody or fragmentthereof into a cell culture medium of the invention during cell culture.Accordingly, compositions of the invention may comprise a mammalian cellthat produces bevacizumab or fragment thereof and a cell culture mediumprovided herein into which the bevacizumab or fragment thereof issecreted. Compositions comprising bevacizumab or fragment thereof and acell culture medium provided herein are also contemplated. In someaspects of the invention, a composition comprises (a) a mammalian cellcomprising a nucleic acid encoding bevacizumab or fragment thereof; and(b) a cell culture medium as provided herein. In some aspects, thecomposition comprises (a) bevacizumab or fragment thereof; and (b) acell culture medium as provided herein, wherein the antibody or fragmentthereof is secreted into the medium by a mammalian cell comprising anisolated nucleic acid encoding bevacizumab or fragment thereof. In otheraspects, the composition comprises: (a) bevacizumab or fragment thereof;and (b) a cell culture medium as provided herein, wherein thebevacizumab or fragment thereof is released into the medium by lysis ofa mammalian cell comprising an isolated nucleic acid encoding thebevacizumab or fragment thereof. The mammalian cell of the compositionmay be any mammalian cell detailed herein (e.g., a CHO cell) and themedium of the composition may be any medium detailed herein, such as amedium comprising one or more compounds as detailed in Table 1.

Compositions (e.g., pharmaceutical formulations) of bevacizumab or afragment thereof produced by any of the methods described herein areprepared by mixing bevacizumab or fragment thereof having the desireddegree of purity with one or more optional pharmaceutically acceptablecarriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.(1980)), which may be in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers, antioxidants, preservatives, lowmolecular weight (less than about 10 residues) polypeptides, proteins;hydrophilic polymers; amino acids; monosaccharides, disaccharides, andother carbohydrates, chelating agents, sugars, salt-formingcounter-ions, metal complexes (e.g. Zn-protein complexes), and/ornon-ionic surfactants. In some embodiments, the pharmaceuticalformulation is administered to a mammal such as a human. Pharmaceuticalformulations of bevacizumab or fragment thereof can be administered byany suitable means, including parenteral, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Dosingcan be by any suitable route, e.g. by injections, such as intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic. Accordingly, antibody-containing formulations asprovided herein may be suitable for injection, such as subcutaneousinjection into an individual (e.g., subcutaneous injection into ahuman). The pharmaceutical formulations to be used for in vivoadministration are generally sterile. Sterility may be readilyaccomplished, for example by filtration through sterile filtrationmembranes.

In some aspects, a composition (e.g., pharmaceutical formulation) asprovided herein comprises bevacizumab or fragment thereof at aconcentration of at least 100 mg/mL, 125 mg/mL, 150 mg/mL, 200 mg/mL, or250 mg/mL, or at a concentration of about 100 mg/mL, about 125 mg/mL,about 150 mg/mL, about 175 mg/mL, or about 200 mg/mL. In other aspects,a composition (e.g., pharmaceutical formulation) as provided hereincomprises bevacizumab or fragment thereof at a concentration of at least1 mg/mL, 10 mg/mL, 25 mg/mL, 50 mg/mL, or 75 mg/mL, or at aconcentration of about 1 mg/mL, about 10 mg/mL, about 25 mg/mL, about 50mg/mL, or about 75 mg/mL.

V. Articles of Manufacture and Kits

A kit for supplementing a cell culture medium with at least two ofcopper, insulin and cystine are provided. The at least two of copper,insulin and cystine may be present in an amount to provide aconcentration of the components at provided in Table 1. The kit maycontain dried constituents to be reconstituted, and may also containinstructions for use (e.g., for use in supplementing a medium with thekit constituents). The kit may contain the constituents provided hereinin amounts suitable to supplement a cell culture medium for use inculturing a mammalian cell comprising a nucleic acid encodingbevacizumab or fragment thereof. In one aspect, a kit comprises cystinein an amount to provide from about 0.9 mM to about 1.5 mM cystine in thecell culture medium. In some embodiments herein, the kit furthercomprises insulin in an amount to provide from about 1.4 mg/L to about11 mg/L insulin in the cell culture medium. In some embodiments herein,the kit further comprises copper in an amount to provide from about 26nM to about 400 nM copper in the cell culture medium._In someembodiments, a kit comprises two or more constituents selected from thegroup consisting of insulin in an amount to provide from about 7.0 mg/Lto about 11.0 mg/L insulin in the cell culture medium, cystine in anamount to provide from about 0.8 mM to about 2.5 mM cystine in the cellculture medium, and copper in an amount to provide from about 25.0 nM toabout 400.0 nM copper in the cell culture medium.

In any of the aspects herein, the kit may further comprise ananimal-derived hydrolysate or a plant-derived hydrolysate or both ananimal-derived hydrolysate and a plant-derived hydrolysate. In some ofthe embodiments herein, the kit further comprises a plant-derivedhydrolysate in an amount to provide from about 1.4 g/L to about 6.2 g/Lplant-derived hydrolysate in the cell culture medium. In some of theembodiments herein, the kit further comprises an animal-derivedhydrolysate in an amount to provide from about 5.6 g/L to about 38.0 g/Lanimal-derived hydrolysate in the cell culture medium.

In another aspect of the invention, an article of manufacture isprovided comprising a container which holds the cell culture medium ofthe invention and optionally provides instructions for its use. Suitablecontainers include, for example, bottles and bags. The container may beformed from a variety of materials such as glass or plastic. Thecontainer holds the cell culture medium and the label on, or associatedwith, the container may indicate directions for use (e.g., for use inculturing cells). The article of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents and package inserts with instructions for use.

An article of manufacture comprising a container which holdsbevacizumab, or a fragment thereof produced by a method detailed hereinand optionally provides instructions for its use is also provided.

EMBODIMENTS

Various embodiments and aspects of the invention are detailed herein andthroughout. Embodiments include, without limitation, the following:

Method 1: A method of producing bevacizumab, or a fragment thereof,comprising the step of culturing a mammalian cell comprising a nucleicacid encoding bevacizumab or fragment thereof in a cell culture medium,wherein the cell culture medium comprises two or more componentsselected from the group consisting of copper, insulin, and cystine, andwherein the cell produces bevacizumab, or a fragment thereof.

Method 2: A method of culturing a mammalian cell comprising a nucleicacid encoding bevacizumab, or a fragment thereof, the method comprisingthe step of contacting the mammalian cell with a cell culture mediumcomprising two or more components selected from the group consisting ofcopper, insulin and cystine.

Method 3: A method of enhancing the amount of bevacizumab, or a fragmentthereof, produced from a mammalian cell comprising a nucleic acidencoding bevacizumab, or a fragment thereof, the method comprising thestep of culturing the mammalian cell in a cell culture medium comprisingat least two of insulin, copper and cystine, wherein the amount ofbevacizumab, or a fragment thereof, produced from the mammalian cell isenhanced relative to culturing the mammalian cell in a cell culturemedium without at least two of insulin, copper and cystine.

Method 4: A method of culturing a mammalian cell comprising a nucleicacid encoding bevacizumab, or a fragment thereof, in a cell culturemedium comprising at least two of insulin, copper and cystine, whereinthe amount of bevacizumab, or a fragment thereof, produced from themammalian cell is enhanced relative to culturing the mammalian cell in acell culture medium without at least two of insulin, copper and cystine.

For any of methods 1-4, the method may comprise any one or more of thefollowing features (i)-(xviii) or sub-feature thereof or any combinationof feature or sub feature:

-   -   (i) the cell culture medium comprises copper and insulin    -   (ii) the cell culture medium comprises copper and cystine    -   (iii) the cell culture medium comprises insulin and cystine    -   (iv) the cell culture medium comprises copper, insulin, and        cystine    -   (v) the cell culture medium (including a medium containing any        of features (i)-(iv)) further comprises a plant-derived        hydrolysate, an animal-derived hydrolysate or both a        plant-derived hydrolysate and an animal-derived hydrolysate.    -   (vi) the cell culture medium (including a medium containing any        one or more or all of features (i)-(v)) comprises insulin at a        concentration of any one of:        -   a. from about 1.0 mg/L to about 100.0 mg/L        -   b. from about 10.0 mg/L to about 100.0 mg/L        -   c. from about 10.0 mg/L to about 50.0 mg/L        -   d. from about 10.0 mg/L to about 35.0 mg/L        -   e. from about 10.0 mg/L to about 25.0 mg/L        -   f. from about 5.0 mg/L to about 80.0 mg/L        -   g. from about 5.0 mg/L to about 60.0 mg/L        -   h. from about 5.0 mg/L to about 50.0 mg/L        -   i. from about 5.0 mg/L to about 40.0 mg/L        -   j. from about 5.0 mg/L to about 25.0 mg/L        -   k. from about 10.0 mg/L to about 25.0 mg/L        -   l. from about 10.0 mg/L to about 40.0 mg/L        -   m. from about 15.0 mg/L to about 20.0 mg/L        -   n. from about 5.0 mg/L to about 15.0 mg/L        -   o. from about 6.0 mg/L to about 12.0 mg/L        -   p. from about 7.0 mg/L to about 11.0 mg/L        -   q. from about 8.0 mg/L to about 10.0 mg/L        -   r. about any one of 5.0 mg/L, 6.0 mg/L, 7.0 mg/L, 8.0 mg/L,            9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0 mg/L, 13.0 mg/L, 14.0            mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0 mg/L, 19.0 mg/L,            20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0 mg/L, 25.0            mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0            mg/L or 31.0 mg/L or 32 mg/L or 33 mg/L or 34 mg/L or 35            mg/L or 36 mg/L or 37 mg/L or 38 mg/L or 39 mg/L or 40 mg/L        -   s. about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L or            11.0 mg/L        -   t. about 25 mg/L    -   (vii) the cell culture medium (including a medium containing any        one or more or all of features (i)-(vi)) comprises copper at a        concentration of any one of:        -   a. from about 69 nM to about 1,000 nM        -   b. from about 69.0 nM to about 400.0 nM        -   c. from about 80 nM to about 400 nM.        -   d. from about 100 nM to about 400 nM        -   e. from about 125 nM to about 400 nM        -   f. from about 150 nM to about 400 nM        -   g. from about 200 nM to about 400 nM        -   h. from about 250 nM to about 400 nM        -   i. from about 300 nM to about 400 nM        -   j. from about 325 nM to about 375 nM        -   k. from about 325 nM to about 350 nM        -   l. about any one of 100 nM, 125 nM, 150 nM, 175 nM, 200 nM,            225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM or            400 nM.        -   m. about any one of 330 nM, 335 nM, 340 nM, 345 nM or 350 nM        -   n. about 335 nM, 336 nM, 337 nM, 338 nM, 339 nM or 400 nM        -   o. about 339 nM    -   (viii) the cell culture medium (including a medium containing        any one or more or all of features (i)-(vii)) comprises cystine        at a concentration of any one of:        -   a. from about 0.7 mM to about 2.0 mM        -   b. from about 0.8 mM to about 2.5 mM        -   c. from about 0.8 mM to about 2.0 mM        -   d. from about 0.8 mM to about 1.75 mM        -   e. from about 0.8 mM to about 1.6 mM        -   f. from about 1.0 mM to about 2.0 mM        -   g. from about 1.0 mM to about 1.6 mM        -   h. from about 1.2 mM to about 1.4 mM        -   i. about any one of 0.8 mM or 0.9 mM or 1.0 mM or 1.1 mM or            1.2 mM or 1.3 mM or 1.4 mM or 1.5 mM        -   j. about any one of 1.1 mM, 1.3 mM or 1.5 mM    -   (ix) the cell culture medium (including a medium containing any        one or more or all of features (i)-(viii)) comprises an        animal-derived hydrolysate at a concentration of any one of:        -   a. from about 6.0 g/L to about 20.0 g/L        -   b. from about 5.6 g/L to about 38.0 g/L        -   c. from about 7.0 g/L to about 25.0 g/L        -   d. from about 7.0 g/L to about 20.0 g/L        -   e. from about 7.0 g/L to about 15.0 g/L        -   f. from about 8.0 g/L to about 12.0 g/L        -   g. from about 9.0 g/L to about 11.0 g/L        -   h. from about 7.0 g/L to about 11.0 g/L        -   i. about any one of 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L,            30 g/L, 35 g/L, 40 g/L, 45 g/L or 50 g/L        -   j. about any one of 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10            g/L, 11 g/L, or 12 g/L        -   k. about 10 g/L        -   l. about 13 g/L    -   (x) the cell culture medium (including a medium containing any        one or more or all of features (i)-(ix)) comprises a        plant-derived hydrolysate at a concentration of any one of:        -   a. from about 1.0 g/L to about 10.0 g/L        -   b. from about 1.4 g/L to about 11.0 g/L        -   c. from about 1.4 g/L to about 6.2 g/L        -   d. from about 1.5 g/L to about 5.5 g/L        -   e. from about 1.5 g/L to about 4.5 g/L        -   f. from about 1.5 g/L to about 3.5 g/L        -   g. from about 2.0 g/L to about 3.0 g/L        -   h. from about 1.5 g/L to about 2.5 g/L        -   i. from about 1.75 g/L to about 2.75 g/L        -   j. from about 2.25 g/L to about 2.75 g/L        -   k. about any one of 1.75 g/L, 2.0 g/L, 2.25 g/L, 2.5 g/L,            3.0 g/L, 3.25, 3.5 g/L, 3.75 g/L, or 4.0 g/L        -   l. about any one of 2.0 g/L, 2.25 g/L, 2.5 g/L or 3.0 g/L        -   m. about 2.5 g/L        -   n. about 3.1 g/L    -   (xi) the cell culture medium (including a medium containing any        one or more or all of features (i)-(x)) comprises both an        animal-derived hydrolysate and a plant-derived hydrolysate, and        wherein the animal-derived hydrolysate is present in a greater        amount than the plant-derived hydrolysate    -   (xii) the cell culture medium (including a medium containing any        one or more or all of features (i)-(xi)) comprises insulin and        the method further comprises the step of adding an additional        amount of insulin to the cell culture medium, wherein the        additional amount of insulin may: (a) be added to the cell        culture medium once or at least three times or at least six        times during the cell culture cycle and (b) may be added in an        amount to provide insulin in the cell culture medium at a        concentration of any one of:        -   a. from about 1.0 mg/L to about 100.0 mg/L        -   b. from about 10.0 mg/L to about 100.0 mg/L        -   c. from about 10.0 mg/L to about 50.0 mg/L        -   d. from about 10.0 mg/L to about 35.0 mg/L        -   e. from about 10.0 mg/L to about 25.0 mg/L        -   f. from about 5.0 mg/L to about 80.0 mg/L        -   g. from about 5.0 mg/L to about 60.0 mg/L        -   h. from about 5.0 mg/L to about 50.0 mg/L        -   i. from about 5.0 mg/L to about 40.0 mg/L        -   j. from about 5.0 mg/L to about 25.0 mg/L        -   k. from about 10.0 mg/L to about 25.0 mg/L        -   l. from about 10.0 mg/L to about 40.0 mg/L        -   m. from about 15.0 mg/L to about 20.0 mg/L        -   n. from about 5.0 mg/L to about 15.0 mg/L        -   o. from about 6.0 mg/L to about 12.0 mg/L        -   p. from about 7.0 mg/L to about 11.0 mg/L        -   q. from about 8.0 mg/L to about 10.0 mg/L        -   r. about any one of 5.0 mg/L, 6.0 mg/L, 7.0 mg/L, 8.0 mg/L,            9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0 mg/L, 13.0 mg/L, 14.0            mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0 mg/L, 19.0 mg/L,            20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0 mg/L, 25.0            mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0            mg/L or 31.0 mg/L or 32 mg/L or 33 mg/L or 34 mg/L or 35            mg/L or 36 mg/L or 37 mg/L or 38 mg/L or 39 mg/L or 40 mg/L        -   s. about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L or            11.0 mg/L        -   t. about 25 mg/L        -   u. about 15 mg/L    -   (xiii) the method further comprises the step of adding cysteine        to the cell culture medium (including a medium containing any        one or more or all of features (i)-(xii)), which cysteine may be        added to the cell culture medium (a) as a component of a batch        feed that is added to a basal medium that does not comprise        cysteine and/or (b) may be added in an amount to provide        cysteine in the cell culture medium at a concentration of from        about 0.5 to about 5.0 mM or from about 0.5 to about 2.0 mM or        from about 0.5 to about 2.0 mM (such as at a concentration of        0.8 mM) or from about 7.0 to about 8.0 mM (such as at a        concentration of about 7.5 mM)    -   (xiv) the method further comprises the step of adding cystine to        the cell culture medium (including a medium containing any one        or more or all of features (i)-(xii)), which cystine may be        added to the cell culture medium as a component of a batch feed        that is added to a basal medium and which may be added in an        amount to provide cystine in the cell culture medium at a        concentration of from about 0.1 to about 1.5 mM (such as at a        concentration of 0.2 mM)    -   (xv) the cell is cultured (e.g., in any cell culture media        including those having any one or more or all of features        (i)-(xiv)) at a temperature ranging from about 28° C. to about        37° C. or from about 31° C. to about 35° C.    -   (xvi) the cell is cultured (e.g., in any cell culture media        including those having any one or more or all of features        (i)-(xiv)) at a first temperature of about 35° C. for a first        period of time, is cultured at a second temperature of about        33° C. for a second period of time, and is cultured at a third        temperature of about 31° C. for a third period of time    -   (xvii) bevacizumab, or a fragment thereof, is secreted into the        cell culture medium (including a medium containing any one or        more or all of features (i)-(xvi))    -   (xviii) the method further comprises the step of recovering the        bevacizumab, or a fragment thereof, from the cell culture        (including a medium containing any one or more or all of        features (i)-(xvi))

Also provided herein is bevacizumab, or fragment thereof, produced byany method provided herein, including without limitation any of methods1-4, which method may further comprise any one or more or all of thefeatures (i)-(xviii) or sub-feature thereof or any combination of theforegoing.

Also provided is a composition comprising: (i) bevacizumab, or afragment thereof, produced by any method provided herein, includingwithout limitation any of methods 1-4, which method may further compriseany one or more or all of the features (i)-(xviii) or sub-featurethereof or any combination of the foregoing and (ii) a pharmaceuticallyacceptable carrier.

A kit for supplementing a cell culture medium for use in culturing amammalian cell comprising a nucleic acid encoding bevacizumab, or afragment thereof, is also provided, the kit comprising at least two ofcomponents (i)-(iii):

-   -   (i) insulin in an amount to provide a concentration of any one        of:        -   a. from about 1.0 mg/L to about 100.0 mg/L        -   b. from about 10.0 mg/L to about 100.0 mg/L        -   c. from about 10.0 mg/L to about 50.0 mg/L        -   d. from about 10.0 mg/L to about 35.0 mg/L        -   e. from about 10.0 mg/L to about 25.0 mg/L        -   f. from about 5.0 mg/L to about 80.0 mg/L        -   g. from about 5.0 mg/L to about 60.0 mg/L        -   h. from about 5.0 mg/L to about 50.0 mg/L        -   i. from about 5.0 mg/L to about 40.0 mg/L        -   j. from about 5.0 mg/L to about 25.0 mg/L        -   k. from about 10.0 mg/L to about 25.0 mg/L        -   l. from about 10.0 mg/L to about 40.0 mg/L        -   m. from about 15.0 mg/L to about 20.0 mg/L        -   n. from about 5.0 mg/L to about 15.0 mg/L        -   o. from about 6.0 mg/L to about 12.0 mg/L        -   p. from about 7.0 mg/L to about 11.0 mg/L        -   q. from about 8.0 mg/L to about 10.0 mg/L        -   r. about any one of 5.0 mg/L, 6.0 mg/L, 7.0 mg/L, 8.0 mg/L,            9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0 mg/L, 13.0 mg/L, 14.0            mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0 mg/L, 19.0 mg/L,            20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0 mg/L, 25.0            mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0            mg/L or 31.0 mg/L or 32 mg/L or 33 mg/L or 34 mg/L or 35            mg/L or 36 mg/L or 37 mg/L or 38 mg/L or 39 mg/L or 40 mg/L        -   s. about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L or            11.0 mg/L.        -   t. about 25 mg/L    -   (ii) cystine in an amount to provide a concentration of any one        of:        -   a. from about 0.7 mM to about 2.0 mM        -   b. from about 0.8 mM to about 2.5 mM        -   c. from about 0.8 mM to about 2.0 mM        -   d. from about 0.8 mM to about 1.75 mM        -   e. from about 0.8 mM to about 1.6 mM        -   f. from about 1.0 mM to about 2.0 mM        -   g. from about 1.0 mM to about 1.6 mM        -   h. from about 1.2 mM to about 1.4 mM        -   i. about any one of 0.8 mM or 0.9 mM or 1.0 mM or 1.1 mM or            1.2 mM or 1.3 mM or 1.4 mM or 1.5 mM        -   j. about any one of 1.1 mM, 1.3 mM or 1.5 mM    -   (iii) copper at a concentration of any one of:        -   a. from about 69.0 nM to about 1,000.0 nM        -   b. from about 69.0 nM to about 400.0 nM        -   c. from about 80 nM to about 400 nM.        -   d. from about 100 nM to about 400 nM        -   e. from about 125 nM to about 400 nM        -   f. from about 150 nM to about 400 nM        -   g. from about 200 nM to about 400 nM        -   h. from about 250 nM to about 400 nM        -   i. from about 300 nM to about 400 nM        -   j. from about 325 nM to about 375 nM        -   k. from about 325 nM to about 350 nM        -   l. about any one of 100 nM, 125 nM, 150 nM, 175 nM, 200 nM,            225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM or            400 nM.        -   m. about any one of 330 nM, 335 nM, 340 nM, 345 nM or 350 nM        -   n. about 335 nM, 336 nM, 337 nM, 338 nM, 339 nM or 400 nM        -   o. about 339 nM

The kit may further comprise other components, including any one or moreof:

-   -   i. an animal-derived hydrolysate, such as in an amount to        provide concentration of any one of:        -   a. from about 6.0 g/L to about 20.0 g/L        -   b. from about 5.6 g/L to about 38.0 g/L        -   c. from about 7.0 g/L to about 25.0 g/L        -   d. from about 7.0 g/L to about 20.0 g/L        -   e. from about 7.0 g/L to about 15.0 g/L        -   f. from about 8.0 g/L to about 12.0 g/L        -   g. from about 9.0 g/L to about 11.0 g/L        -   h. from about 7.0 g/L to about 11.0 g/L        -   i. about any one of 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L,            30 g/L, 35 g/L, 40 g/L, 45 g/L or 50 g/L        -   j. about any one of 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10            g/L, 11 g/L, or 12 g/L        -   k. about 10 g/L        -   l. about 13 g/L    -   ii. plant-derived hydrolysate, such as in an amount to provide a        concentration of any one of:        -   a. from about 1.0 g/L to about 10.0 g/L        -   b. from about 1.4 g/L to about 11.0 g/L        -   c. from about 1.4 g/L to about 6.2 g/L        -   d. from about 1.5 g/L to about 5.5 g/L        -   e. from about 1.5 g/L to about 4.5 g/L        -   f. from about 1.5 g/L to about 3.5 g/L        -   g. from about 2.0 g/L to about 3.0 g/L        -   h. from about 1.5 g/L to about 2.5 g/L        -   i. from about 1.75 g/L to about 2.75 g/L        -   j. from about 2.25 g/L to about 2.75 g/L        -   k. about any one of 1.75 g/L, 2.0 g/L, 2.25 g/L, 2.5 g/L,            3.0 g/L, 3.25, 3.5 g/L, 3.75 g/L, or 4.0 g/L        -   l. about any one of 2.0 g/L, 2.25 g/L, 2.5 g/L or 3.0 g/L        -   m. about 2.5 g/L        -   n. about 3.1 g/L

A cell culture medium for use in culturing a mammalian cell comprising anucleic acid encoding bevacizumab, or a fragment thereof, is alsoprovided, the cell culture medium comprising at least two of components(i)-(iii):

-   -   (i) insulin in an amount to provide a concentration of any one        of:        -   a. from about 1.0 mg/L to about 100.0 mg/L        -   b. from about 10.0 mg/L to about 100.0 mg/L        -   c. from about 10.0 mg/L to about 50.0 mg/L        -   d. from about 10.0 mg/L to about 35.0 mg/L        -   e. from about 10.0 mg/L to about 25.0 mg/L        -   f. from about 5.0 mg/L to about 80.0 mg/L        -   g. from about 5.0 mg/L to about 60.0 mg/L        -   h. from about 5.0 mg/L to about 50.0 mg/L        -   i. from about 5.0 mg/L to about 40.0 mg/L        -   j. from about 5.0 mg/L to about 25.0 mg/L        -   k. from about 10.0 mg/L to about 25.0 mg/L        -   l. from about 10.0 mg/L to about 40.0 mg/L        -   m. from about 15.0 mg/L to about 20.0 mg/L        -   n. from about 5.0 mg/L to about 15.0 mg/L        -   o. from about 6.0 mg/L to about 12.0 mg/L        -   p. from about 7.0 mg/L to about 11.0 mg/L        -   q. from about 8.0 mg/L to about 10.0 mg/L        -   r. about any one of 5.0 mg/L, 6.0 mg/L, 7.0 mg/L, 8.0 mg/L,            9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0 mg/L, 13.0 mg/L, 14.0            mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0 mg/L, 19.0 mg/L,            20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0 mg/L, 25.0            mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0            mg/L or 31.0 mg/L or 32 mg/L or 33 mg/L or 34 mg/L or 35            mg/L or 36 mg/L or 37 mg/L or 38 mg/L or 39 mg/L or 40 mg/L        -   s. about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L or            11.0 mg/L        -   t. about 25 mg/L    -   (ii) cystine in an amount to provide a concentration of any one        of:        -   a. from about 0.7 mM to about 2.0 mM        -   b. from about 0.8 mM to about 2.5 mM        -   c. from about 0.8 mM to about 2.0 mM        -   d. from about 0.8 mM to about 1.75 mM        -   e. from about 0.8 mM to about 1.6 mM        -   f. from about 1.0 mM to about 2.0 mM        -   g. from about 1.0 mM to about 1.6 mM        -   h. from about 1.2 mM to about 1.4 mM        -   i. about any one of 0.8 mM or 0.9 mM or 1.0 mM or 1.1 mM or            1.2 mM or 1.3 mM or 1.4 mM or 1.5 mM        -   j. about any one of 1.1 mM, 1.3 mM or 1.5 mM    -   (iii) copper at a concentration of any one of:        -   a. from about 69.0 nM to about 1,000.0 nM        -   b. from about 69.0 nM to about 400.0 nM        -   c. from about 80 nM to about 400 nM.        -   d. from about 100 nM to about 400 nM        -   e. from about 125 nM to about 400 nM        -   f. from about 150 nM to about 400 nM        -   g. from about 200 nM to about 400 nM        -   h. from about 250 nM to about 400 nM        -   i. from about 300 nM to about 400 nM        -   j. from about 325 nM to about 375 nM        -   k. from about 325 nM to about 350 nM        -   l. about any one of 100 nM, 125 nM, 150 nM, 175 nM, 200 nM,            225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM or            400 nM.        -   m. about any one of 330 nM, 335 nM, 340 nM, 345 nM or 350 nM        -   n. about 335 nM, 336 nM, 337 nM, 338 nM, 339 nM or 400 nM        -   o. about 339 nM

The cell culture medium may comprise other components, including any oneor more of:

-   -   1) an animal-derived hydrolysate, such as in an amount to        provide concentration of any one of:        -   a. from about 6.0 g/L to about 20.0 g/L        -   b. from about 5.6 g/L to about 25.0 g/L        -   c. from about 7.0 g/L to about 25.0 g/L        -   d. from about 7.0 g/L to about 20.0 g/L        -   e. from about 7.0 g/L to about 15.0 g/L        -   f. from about 8.0 g/L to about 12.0 g/L        -   g. from about 9.0 g/L to about 11.0 g/L        -   h. from about 7.0 g/L to about 11.0 g/L        -   i. about any one of 5 g/L, 10 g/L, 15 g/L, 20 g/L or 25 g/L        -   j. about any one of 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10            g/L, 11 g/L, or 12 g/L        -   k. about 10 g/L        -   l. about 13 g/L    -   2) plant-derived hydrolysate, such as in an amount to provide a        concentration of any one of:        -   a. from about 1.0 g/L to about 10.0 g/L        -   b. from about 1.4 g/L to about 11.0 g/L        -   c. from about 1.4 g/L to about 6.2 g/L        -   d. from about 1.5 g/L to about 5.5 g/L        -   e. from about 1.5 g/L to about 4.5 g/L        -   f. from about 1.5 g/L to about 3.5 g/L        -   g. from about 2.0 g/L to about 3.0 g/L        -   h. from about 1.5 g/L to about 2.5 g/L        -   i. from about 1.75 g/L to about 2.75 g/L        -   j. from about 2.25 g/L to about 2.75 g/L        -   k. about any one of 1.75 g/L, 2.0 g/L, 2.25 g/L, 2.5 g/L,            3.0 g/L, 3.25, 3.5 g/L, 3.75 g/L, or 4.0 g/L        -   l. about any one of 2.0 g/L, 2.25 g/L, 2.5 g/L or 3.0 g/L        -   m. about 2.5 g/L        -   n. about 3.1 g/L

The cell culture medium may further be supplemented with additional cellculture medium components, where the additional cell culture mediumcomponents may comprise, e.g., insulin and/or cysteine, such as insulinin an amount to provide any concentration of insulin provided herein,including the concentrations listed in the present embodiments (such as15 mg/L) and/or cysteine in an amount to provide any concentration ofcysteine provided herein (such as 0.8 mM), including the concentrationslisted in the present embodiments.

The cell culture medium may further be supplemented with additional cellculture medium components, where the additional cell culture mediumcomponents may comprise, e.g., insulin and/or cysteine and/or cystine,such as insulin in an amount to provide any concentration of insulinprovided herein, including the concentrations listed in the presentembodiments (such as 15 mg/L) and/or cysteine in an amount to provideany concentration of cysteine provided herein (such as 0.8 mM),including the concentrations listed in the present embodiments and/orcystine in an amount to provide any concentration of cystine providedherein (such as 0.2 mM), including the concentrations listed in thepresent embodiments.

Also provided herein is a composition comprising (a) a mammalian cellcomprising a nucleic acid encoding bevacizumab, or a fragment thereof;and (b) a cell culture medium detailed herein, including withoutlimitation a cell culture medium provided in the present embodiments.Further provided is a composition comprising: (a) bevacizumab, or afragment thereof; and (b) a cell culture medium detailed herein,including without limitation a cell culture medium provided in thepresent embodiments.

Also provided herein is a method of producing bevacizumab or a fragmentthereof, comprising a step of culturing a mammalian cell comprising anucleic acid encoding bevacizumab or a fragment thereof in a cellculture medium, wherein initial cell culture medium in a cell culturecycle comprises two or more components selected from the groupconsisting of copper at a concentration of from about 69 nM to about1,000 nM, insulin at a concentration of from about 1.0 mg/L to about100.0 mg/L, and cystine at a concentration of from about 0.7 mM to about2.0 mM, and wherein the cell produces bevacizumab or the fragment.

In some embodiments, without limitation, the method may comprise any oneor more of the following features (i)-(xxiii) or sub-feature thereof orany combination of feature or sub feature:

-   -   (i) the initial cell culture medium comprises copper and insulin    -   (ii) the initial cell culture medium comprises copper and        cystine    -   (iii) the initial cell culture medium comprises insulin and        cystine    -   (iv) the initial cell culture medium comprises copper, insulin,        and cystine    -   (v) the initial cell culture medium (including a medium        containing any one or more or all of features (i)-(iv))        comprises insulin at a concentration of any one of:        -   a. from about 10.0 mg/L to about 50.0 mg/L        -   b. from about 10.0 mg/L to about 20.0 mg/L        -   c. about any one of 10.0 mg/L, 15.0 mg/L, 20.0 mg/L, and            25.0 mg/L    -   (vi) the initial cell culture medium (including a medium        containing any one or more or all of features (i)-(v)) comprises        copper at a concentration of any one of:        -   a. from about 325 nM to about 375 nM        -   b. from about 325 nM to about 350 nM        -   c. about any one of 330 nM, 335 nM, 339 nM, 340 nM, 345 nM            and 350 nM    -   (vii) the initial cell culture medium (including a medium        containing any one or more or all of features (i)-(vi))        comprises cystine at a concentration of any one of:        -   a. from about 0.7 mM to about 2.0 mM        -   b. from about 1.0 mM to about 1.6 mM        -   c. about any one of 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM 1.4 mM,            1.5 mM and 1.6 mM    -   (viii) the initial cell culture medium (including a medium        containing any of features (i)-(vii)) further comprises a        plant-derived hydrolysate, an animal-derived hydrolysate or both        a plant-derived hydrolysate and an animal-derived hydrolysate    -   (ix) the initial cell culture medium (including a medium        containing any one or more or all of features (i)-(viii))        comprises an animal-derived hydrolysate at a concentration of        any one of:        -   a. from about 6.0 g/L to about 20.0 g/L        -   b. from about 8.0 g/L to about 12.0 g/L        -   c. from about 9.0 g/L to about 11.0 g/L        -   d. about 13 g/L    -   (x) the initial cell culture medium (including a medium        containing any one or more or all of features (i)-(ix))        comprises a plant-derived hydrolysate at a concentration of any        one of:        -   a. from about 1.0 g/L to about 10.0 g/L        -   b. from about 2.0 g/L to about 3.0 g/L        -   c. from about 2.25 g/L to about 2.75 g/L        -   d. about 2.5 g/L    -   (xi) the initial cell culture medium (including a medium        containing any one or more or all of features (i)-(x)) comprises        both an animal-derived hydrolysate and a plant-derived        hydrolysate, and wherein the animal-derived hydrolysate is        present in a greater amount than the plant-derived hydrolysate    -   (xii) the initial cell culture medium (including a medium        containing any one or more or all of features (i)-(xi))        comprises insulin and the method further comprises the step of        adding an additional amount of insulin to the cell culture        medium during the cell culture cycle, and optionally wherein a)        the additional amount of insulin may be added to the cell        culture medium at least once, at least twice, at least three        times, at least four times, at least five times, or at least six        times during the cell culture cycle; and/or b) the insulin added        each time is any one of: from about 5.0 mg/L to about 25.0 mg/L        or about any one of 5.0 mg/L, 10.0 mg/L, 15.0 mg/L, 20.0 mg/L,        and 25.0 mg/L; and/or c) the cumulative amount of insulin added        during the cell culture cycle is any one of: from about 20.0        mg/L to about 100.0 mg/L or about any one of 20.0 mg/L, 25.0        mg/L, 30 mg/L, 35 mg/L, 40 mg/L, 45 mg/L, 50 mg/L, 55 mg/L, 60        mg/L, 65 mg/L, 70 mg/L, 75 mg/L, 80 mg/L and 85 mg/L    -   (xiii) the initial cell culture medium (including a medium        containing any one or more or all of features (i)-(xii))        comprises cystine and the method further comprises a step of        adding an additional amount of cystine to the cell culture        medium during the cell culture cycle, and optionally wherein a)        cystine is added in an amount to provide any one of: from about        0.1 mM to about 1.5 mM additional cystine in the cell culture        medium or from about 0.4 mM to about 0.7 mM (e.g., about 0.4 mM        to about 0.6 mM, about 0.4 mM to about 0.5 mM) additional        cystine in the cell culture medium; and/or b) the cystine is        added in a batch feed during the cell culture cycle    -   (xiv) the method further comprises the step of adding any one        of: at least one batch feed during the cell culture cycle, or        two, three, or four batch feeds during the cell culture cycle;        and optionally wherein the batch feed medium may comprise a        plant-derived hydrolysate, an animal-derived hydrolysate or both        a plant-derived hydrolysate and an animal-derived hydrolysate    -   (xv) during the cell culture cycle, the temperature of the        medium (including a medium containing any one or more or all of        features (i)-(xiv)) is reduced by at least 2, at least about 3,        at least about 4, or at least about 5 degrees C. relative to the        temperature at the beginning of the culturing    -   (xvi) during the cell culture cycle, the temperature of the        medium (including a medium containing any one or more or all of        features (i)-(xv)) is a) reduced at least once or at least twice        during the cell culture cycle; and/or b) wherein the temperature        is reduced on day 8 and day 10 after the beginning of the        culturing    -   (xvii) the cell is cultured (e.g., in any cell culture media        including those having any one or more or all of features        (i)-(xvi)) at a temperature ranging from about 31° C. to about        35° C.    -   (xviii) the cell is cultured (e.g., in any cell culture media        including those having any one or more or all of features        (i)-(xvii)) at a first temperature of about 35° C. for a first        period of time, is cultured at a second temperature of about        33° C. for a second period of time, and is cultured at a third        temperature of about 31° C. for a third period of time    -   (xix) the cell is cultured in the medium (e.g., in any cell        culture media including those having any one or more or all of        features (i)-(xviii)) having a pH at about 7.0 to about 7.3    -   (xx) the method comprises (a) culturing the cell in an initial        cell culture medium comprising about 10 mg/L insulin, about 325        nM to about 350 nM copper, and about 1.3 mM cystine; (b)        providing a first batch feed and an insulin feed to the cell        culture medium to provide additional insulin at a concentration        of about 15 mg/L on day 3 after the beginning of the culturing;        and (c) providing a second batch feed comprising cystine to the        cell culture medium to provide additional cystine at a        concentration of about 0.4 mM to about 0.7 mM on day 6 after the        beginning of the culturing; wherein the cell is cultured at an        initial temperature of about 35° C., and the temperature is        reduced to about 33° C. on day 8 and is further reduced to about        31° C. on day 10 after the beginning of the culturing    -   (xxi) bevacizumab, or a fragment thereof, is secreted into the        cell culture medium (including a medium containing any one or        more or all of features (i)-(xx)) (xxii) the method further        comprises the step of recovering the bevacizumab, or a fragment        thereof, from the cell culture (including a medium containing        any one or more or all of features (i)-(xxi))    -   (xxiii) the mammalian cell is a Chinese hamster ovary cell

Also provided herein is bevacizumab, or fragment thereof, produced byany method provided herein, which method may further comprise any one ormore or all of the features (i)-(xxiii) or sub-feature thereof or anycombination of the foregoing.

Also provided is a composition comprising: (i) bevacizumab, or afragment thereof, produced by any method provided herein, which methodmay further comprise any one or more or all of the features (i)-(xxiii)or sub-feature thereof or any combination of the foregoing and (ii) apharmaceutically acceptable carrier.

The following Examples are provided to illustrate but not to limit theinvention.

EXAMPLES Example 1: Impact of Cell Culture Medium Components on Amountof Bevacizumab Produced by a Mammalian Cell Line

Chinese hamster ovary (CHO) cells producing bevacizumab were cultured incell culture media containing various amounts of insulin, and the impactof insulin on the amount of bevacizumab produced was assessed.Production of bevacizumab was initiated in cell culture by inoculatingcells in basal medium containing 339 nM copper, 1% animal hydrolysateand 0.25% plant hydrolysate and a batch feed medium was added on day 3over a 14 day cell culture cycle in a bioreactor. The basal cell culturemedium of a control cell culture was supplemented with less than 10 mg/Lof insulin and no additional insulin was given during the cell culturecycle. In two representative experimental cases (case 1 and case 2), theinsulin level in the basal cell culture media was 10 mg/L. Additionalinsulin was added three to six times during the cell culture cycle so asto provide cell culture case 1 and cell culture case 2 with a finalamount of 25 mg/L and 40 mg/L insulin at the end of the 14 day cellculture cycle, respectively (Table A). The cells were cultured at 37° C.on day 1 and the temperature was subsequently shifted down to 35° C. onday 1, 33° C. on day 8, and 31° C. on day 10. Titer improvement wasquantified by percent increase over the control. Total addition ofinsulin during the cell culture cycle from less than 10 mg/L to 25 mg/Lor 40 mg/L led to a titer improvement of about 16% or about 18% ascompared with the control, respectively (Table A).

TABLE A Exemplary insulin addition protocol and results Insulin Insulinaddition addition case 1 case 2 Total insulin 25 mg/L 40 mg/Lconcentration added during a 14 day duration Titer improvement 16% 18%percent increase

CHO cells producing bevacizumab were cultured in cell culture mediacontaining different amounts of hydrolysate from different sources andthe impact of the specific hydrolysate on the amount of bevacizumabproduced was assessed. In two representative protocols, production ofbevacizumab was initiated in cell culture by inoculating cells in basalcell culture medium containing 339 nM copper and a batch feed medium wasadded on day 3 over a 14 day cell culture cycle in a bioreactor. Thebasal cell culture medium for Protocol 1 had 1% animal-derivedhydrolysate without plant-derived hydrolysate while Protocol 2 had basalcell culture medium supplemented with 0.75% animal-derived hydrolysatein combination with 0.25% plant-derived hydrolysate (Table B). Analysisof the antibody titer produced by the two protocols demonstrated thatProtocol 2 provided a 27% increase in the amount of bevacizumab producedfrom the cells as compared to the amount of bevacizumab produced bycells cultured using Protocol 1 (Table B).

TABLE B Exemplary experimental protocols Protocol 1 Protocol 2 Porcinepeptone 1% 0.75% Plant peptone None 0.25% Titer improvement NotApplicable  27% percent increase

Example 2: Impact of Cell Culture Medium Components on Amount ofBevacizumab Produced by a Mammalian Cell Line

CHO cells producing bevacizumab were cultured in basal media with eitherthe amino acid cysteine in the monomer form (Cys, cysteine) or in thedimer form (Cys-Cys, cystine) (Table C). Production of bevacizumab wasinitiated in cell culture by inoculating cells in basal mediumcontaining 339 nM copper, 1% animal hydrolysate and 0.25% planthydrolysate and a batch feed was added on day 3 over a 14 day cellculture cycle in a bioreactor. Insulin levels in the basal media were ata concentration of either less than 10 mg/L or 10 mg/L. Two of theexperimental cell cultures (Cysteine+Insulin and Cystine+Insulin) hadadditional insulin added three times or six times during the cellculture cycle so as to provide a final amount of 25 mg/L or 40 mg/L(Table C). The cells were cultured at 37° C. on day 1 the temperaturewas subsequently shifted down to 35° C. on day 1, 33° C. on day 8, and31° C. on day 10. Titer improvement was quantified by percent increaseover the control. Replacement of cysteine (Cys) by cystine (Cys-Cys)improved titer by 11% over the control. Impact of insulin addition wasalso observed in basal media made using cystine (Cys-Cys).

TABLE C Summary of protocols and results Cysteine Cysteine + Cystine +(Control) Cystine Insulin Insulin Cysteine (Cys) 2.6 mM  0 mM 2.6 mM  0mM concentration in basal media Cystine (Cys-  0 mM 1.3 mM 0 mM 1.3 mM Cys) concentration in basal media Total insulin Less than Less than 25mg/L 40 mg/L concentration 10 mg/L 10 mg/L added during a 14 dayduration Titer Not 11% 11% 14% improvement Applicable percent increase

Example 3: Impact of Insulin Addition on Amount of Bevacizumab Producedby a Mammalian Cell Line

Chinese hamster ovary (CHO) cells producing bevacizumab were cultured incell culture media containing various amounts of insulin, and the impactof insulin addition on the amount of bevacizumab produced was assessed.Production of bevacizumab was initiated in cell culture by inoculatingcells in basal medium. After inoculation, the medium (i.e., initial cellculture medium) contained 300 nM copper sulfate (CuSO4), 1% animalhydrolysate and 0.25% plant hydrolysate, and 1.3 mM cystine. The mediumalso contained 10 mg/L or 20 mg/L of insulin after inoculation (TableD). A batch feed medium was added on day 3 or a two batch feed mediumswere added (one batch feed on day 3 and a second batch feed on day 6)over a 14 day cell culture cycle in a bioreactor. Some cell culturesreceived additional insulin one to six times during the cell culturecycle so as to provide the cell culture with a cumulative amount of 25mg/L to 85 mg/L insulin at the end of the 14 day cell culture cycle(Table D). The cells were cultured at 37° C. on day 1 and thetemperature was subsequently shifted down to 35° C. on day 1, 33° C. onday 8, and 31° C. on day 10. Titer improvement was compared across thedifferent cultures in two representative experiments and it wasdetermined that 10 mg/L insulin in the initial cell culture medium withaddition of insulin on day 3 for a cumulative amount of 25 mg/L insulinproduced the highest titer yield (FIG. 1A (one batch feed) and 1B (twobatch feeds)). Cell cultures containing 20 mg/mL insulin in the initialcell culture medium with no additional insulin supplementationdemonstrated comparable titer yields to cultures that had 10 mg/mLinsulin in the initial cell culture medium and additional insulin addedduring the cell culture cycle indicating that insulin in the initialcell culture medium was an important factor for increasing titer yield(Table D, FIGS. 1A and 1B).

TABLE D Exemplary insulin addition protocol Cumulative Insulin ininsulin in initial cell Insulin Day of cell culture Protocol culturemedium feed insulin feed at 14 days 1 20 mg/L None None 20 mg/L 2 10mg/L 15 mg/L Day 3 25 mg/L 3 10 mg/L  5 mg/L Days 3, 6, and 9 25 mg/L 410 mg/L  5 mg/L Days 3, 5, 7, 9, 40 mg/L 11, and 13 5 10 mg/L 15 mg/LDays 3, 6, and 9 55 mg/L 6 10 mg/L 25 mg/L Days 3, 6, and 9 85 mg/L

Example 4: Impact of Additional Batch Media Feed on Amount ofBevacizumab Produced by a Mammalian Cell Line

Four different samples of CHO cells producing bevacizumab were culturedin cell culture media and fed with one or two batch media feeds todetermine the impact on the amount of bevacizumab produced. Productionof bevacizumab was initiated in cell culture by inoculating cells inbasal medium. After inoculation, the medium (i.e., initial cell culturemedium) contained 300 nM copper sulfate (CuSO4), 1% animal hydrolysateand 0.25% plant hydrolysate, and 1.3 mM cystine. In one protocol, abatch feed medium was added on day 3 over a 14 day cell culture cycle ina bioreactor. In a second protocol, a batch feed medium was added on day3 and day 6 over a 14 day cell culture cycle in a bioreactor (Table E).The batch feed medium delivered on day 3 contained 2.5% animalhydrolysate and 0.625% plant hydrolysate and was added into the cellculture in a volume of 125 ml/L. The batch feed medium delivered on day6 contained 7.5 mM cysteine and 7.5 mM cystine and was added into thecell culture in a volume of 90 ml/L. Accordingly, the cell culture hadan increase of 0.62 mM in cystine concentration after the feed. Thecells were cultured at 37° C. on day 1 and the temperature wassubsequently shifted down to 35° C. on day 1, 33° C. on day 8, and 31°C. on day 10. Titer improvement was observed in cell cultures receivinga second feed on day 6 with cultures receiving only one feeddemonstrating an average titer, and the two-feed process led to a titerincrease of up to 0.3 g/L (about 20% titer increase). Measurement ofbiomass accumulation (FIG. 2A) and titer (FIG. 2B) at Day 14 indicatedthat a second feed on day 6 improved yield and that this second feed wasmore significant for cultures with higher cell ages where higher growthand an earlier decline in viability is observed.

TABLE E Exemplary batch feed addition protocol Media Protocol 1 Protocol2 Initial cell 1% Animal hydrolysate 1% Animal hydrolysate culturemedium 0.25% Plant hydrolysate 0.25% Plant hydrolysate 1.3 mM Cystine1.3 mM Cystine 10 mg/L Insulin 10 mg/L Insulin 300 nM CuSO4 300 nM CuSO4Batch Feed 2.5% Animal hydrolysate 2.5% Animal hydrolysate on Day 30.625% Plant hydrolysate 0.625% Plant hydrolysate (provided as 125 mL/L)(provided as 125 mL/L) Batch Feed None 7.5 mM Cysteine on Day 6 7.5 mMCystine (provided as 90 mL/L)

Analysis of the above described results provided for the development ofa new process for culturing CHO cells producing bevacizumab. The newprocess incorporated the use of an initial cell culture mediumcontaining 10 mg/L insulin as well as cystine instead of cysteine, abatch feed on day 3 and day 6 during cell culture and the addition ofinsulin on day 3 of cell culture (Table F, Protocol 2). Addition ofthese components to the cell culture process resulted in a titerincrease up to about 60%. See FIG. 3 and Table F, Protocol 2 as comparedProtocol 1.

TABLE F New cell culture process Media Protocol 1 Protocol 2 Initialcell 1% Animal hydrolysate 1% Animal hydrolysate culture medium 2.6 mMCysteine 0.25% Plant hydrolysate 2 mg/L Insulin 1.3 mM Cystine 10 mg/LInsulin 300 nM CuSO4 Batch Feed 2.5% Animal hydrolysate 2.5% Animalhydrolysate on Day 3 (provided as 125 mL/L) 0.62 5% Plant hydrolysate(provided as 125 mL/L) Insulin Feed None 15 mg/L Insulin on Day 3 BatchFeed None 7.5 mM Cysteine on Day 6 7.5 mM Cystine (provided as 90 mL/L)Temperature 33° C. 35° C. on day 1, 33° C. on day 8, and 31° C. on day10

1-225. (canceled)
 226. A method of producing bevacizumab or a fragment thereof, comprising a step of culturing a mammalian cell comprising a nucleic acid encoding bevacizumab or a fragment thereof in a cell culture medium, wherein initial cell culture medium in a cell culture cycle comprises two or more components selected from the group consisting of copper at a concentration of from about 69 nM to about 1,000 nM, insulin at a concentration of from about 1.0 mg/L to about 100.0 mg/L, and cystine at a concentration of from about 0.7 mM to about 2.0 mM, and wherein the cell produces bevacizumab or the fragment.
 227. The method of claim 226, wherein the initial cell culture medium comprises (1) copper and insulin; (2) copper and cystine; (3) insulin and cystine; or (4) copper, insulin, and cystine.
 228. The method of claim 226, wherein the initial cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 50.0 mg/L.
 229. (canceled)
 230. (canceled)
 231. The method of claim 226, wherein the initial cell culture medium comprises copper at a concentration of from about 325 nM to about 375 nM.
 232. (canceled)
 233. (canceled)
 234. The method of claim 226, wherein the initial cell culture medium comprises cystine at a concentration of from about 0.7 mM to about 2.0 mM. 235-246. (canceled)
 247. The method of claim 226, wherein the initial cell culture medium comprises insulin and the method further comprises a step of adding an additional amount of insulin to the cell culture medium during the cell culture cycle.
 248. (canceled)
 249. The method of claim 247, wherein the insulin added each time is from about 5 mg/L to about 25 mg/L.
 250. (canceled)
 251. The method of claim 247, wherein the cumulative amount of insulin added during the cell culture cycle is from about 20 mg/L to about 100 mg/L.
 252. (canceled)
 253. The method of claim 226, wherein the initial cell culture medium comprises cystine and the method further comprises a step of adding an additional amount of cystine to the cell culture medium during the cell culture cycle.
 254. The method of claim 253, wherein cystine is added in an amount to provide from about 0.1 to about 1.5 mM additional cystine in the cell culture medium.
 255. (canceled)
 256. (canceled)
 257. The method of claim 226, wherein the method further comprises at least one batch feed during the cell culture cycle.
 258. (canceled)
 259. (canceled)
 260. The method of claim 226, wherein during the cell culture cycle, the temperature of the medium is reduced by at least about 2, at least about 3, at least about 4, or at least about 5 degrees C. relative to the temperature at the beginning of the culturing. 261-263. (canceled)
 264. The method of claim 226, wherein the cell is cultured at a first temperature of about 35° C. for a first period of time, is cultured at a second temperature of about 33° C. for a second period of time, and is cultured at a third temperature of about 31° C. for a third period of time.
 265. The method of claim 226, wherein the cell is cultured in the medium having a pH at about 7.0 to about 7.3.
 266. The method of claim 226, wherein the method comprises (a) culturing the cell in an initial cell culture medium comprising about 10 mg/L insulin, about 325 nM to about 350 nM copper, and about 1.3 mM cystine; (b) providing a first batch feed and an insulin feed to the cell culture medium to provide additional insulin at a concentration of about 15 mg/L on day 3 after the beginning of the culturing; and (c) providing a second batch feed comprising cystine to the cell culture medium to provide additional cystine at a concentration of about 0.4 to about 0.7 mM on day 6 after the beginning of the culturing; wherein the cell is cultured at an initial temperature of about 35° C., and the temperature is reduced to about 33° C. on day 8 and is further reduced to about 31° C. on day 10 after the beginning of the culturing.
 267. The method of claim 226, wherein bevacizumab or a fragment thereof is secreted into the cell culture medium.
 268. The method of claim 226, further comprising a step of recovering the bevacizumab or a fragment thereof from the cell culture.
 269. The method of claim 226, wherein the mammalian cell is a Chinese hamster ovary cell.
 270. Bevacizumab or a fragment thereof produced by the method of claim
 226. 271. A composition comprising: (i) bevacizumab or a fragment thereof produced by the method of claim 226 and (ii) a pharmaceutically acceptable carrier. 